Peptides for use in immunotherapy against cancers

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of U.S. patent application Ser. No.16/413,439 filed 15 May 2019, which claims the benefit of U.S.Provisional Application Ser. No. 62/672,411 filed 16 May 2018, andGermany Application No. 10201811819.8, filed 16 May 2018 the content ofeach of these applications is herein incorporated by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_listing_2912919-093003_ST25.txt” createdon 30 Jun. 2021, and 71,498 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

FIELD

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

BACKGROUND OF THE INVENTION

According to the World Health Organization (WHO), cancer ranged amongthe four major non-communicable deadly diseases worldwide in 2012. Forthe same year, colorectal cancer, breast cancer and respiratory tractcancers were listed within the top 10 causes of death in high incomecountries.

Considering the severe side-effects and expenses associated with commonstrategies for treating cancer, there is a need to identify factors thatcan be used in the treatment of cancer in general and acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer inparticular.

There is also a need to identify factors representing biomarkers forcancer in general and acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer in particular, leading to betterdiagnosis of cancer, assessment of prognosis, and prediction oftreatment success.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor-(-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell-(CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Longer (elongated) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach (Singh-Jasuja et al., 2004). It isessential that epitopes are present in the amino acid sequence of theantigen, in order to ensure that such a peptide (“immunogenic peptide”),being derived from a tumor associated antigen, leads to an in vitro orin vivo T-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a T cellbased therapy including but not limited to tumor vaccines. The methodsfor identifying and characterizing the TAAs are usually based on the useof T-cells that can be isolated from patients or healthy subjects, orthey are based on the generation of differential transcription profilesor differential peptide expression patterns between tumors and normaltissues. However, the identification of genes over-expressed in tumortissues or human tumor cell lines, or selectively expressed in suchtissues or cell lines, does not provide precise information as to theuse of the antigens being transcribed from these genes in an immunetherapy. This is because only an individual subpopulation of epitopes ofthese antigens are suitable for such an application since a T cell witha corresponding TCR has to be present and the immunological tolerancefor this particular epitope needs to be absent or minimal. In a verypreferred embodiment of the invention it is therefore important toselect only those over- or selectively presented peptides against whicha functional and/or a proliferating T cell can be found. Such afunctional T cell is defined as a T cell, which upon stimulation with aspecific antigen can be clonally expanded and is able to executeeffector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 387 and SEQ ID NO: 463 toSEQ ID NO: 464 or a variant sequence thereof which is at least 77%,preferably at least 88%, homologous (preferably at least 77% or at least88% identical) to SEQ ID NO: 1 to SEQ ID NO: 387 and SEQ ID NO: 463 toSEQ ID NO: 464, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention also relates to a peptide of the present inventioncomprising a sequence that is selected from the group consisting of SEQID NO: 1 to SEQ ID NO: 387 and SEQ ID NO: 463 to SEQ ID NO: 464 or avariant thereof, which is at least 77%, preferably at least 88%,homologous (preferably at least 77% or at least 88% identical) to SEQ IDNO: 1 to SEQ ID NO: 387 and SEQ ID NO: 463 to SEQ ID NO: 464, whereinsaid peptide or variant thereof has an overall length of between 8 and100, preferably between 8 and 30, and most preferred of between 8 and 14amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. In Table 1a, peptides with SEQ IDNO: 1 to SEQ ID NO: 382 and in Table 1 b, peptides with SEQ ID NO: 463to SEQ ID NO: 464 bind to HLA-A*24. The peptides in Table 2 have beendisclosed before in large listings as results of high-throughputscreenings with high error rates or calculated using algorithms but havenot been associated with cancer at all before. In Table 2, peptides withSEQ ID NO: 383 to SEQ ID NO: 387 bind to HLA-A*24. The peptides in Table3 are additional peptides that may be useful in combination with theother peptides of the invention. In Table 3, peptides with SEQ ID NO:388 to SEQ ID NO: 460 bind to HLA-A*24.

TABLE 1a Peptides according to the present invention. Seq ID HLA NoSequence Gene(s) allotype 1 IFPKTGLLII MAGEA4 A*24 2 LYAPTILLW AFP A*243 KFLTHDVLTELF TRPM8 A*24 4 MVLQPQPQLF POTEG, POTEH A*24 5 LQPQPQLFFSFPOTEG, POTEH A*24 6 IVTFMNKTLGTF ADAM29 A*24 7 GYPLRGSSI ALPP, ALPPL2A*24 8 IMKPLDQDF ADAM2 A*24 9 TYINSLAIL TGM4 A*24 10 QYPEFSIEL TGM4 A*2411 RAMCAMMSF TGM4 A*24 12 KYMSRVLFVY CHRNA9 A*24 13 KYYIATMAL CHRNA9A*24 14 YYIATMALI CHRNA9 A*24 15 FMVIAGMPLF SLC6A3 A*24 16 GYFLAQYLMTRPM8 A*24 17 IYPEAIATL SLC6A3 A*24 18 KYVDINTFRL MMP12 A*24 19ILLCMSLLLF CYP4Z1, CYP4Z2P A*24 20 ELMAHPFLL CYP4Z1, CYP4Z2P A*24 21LYMRFVNTHF SPINK2 A*24 22 VYSSFVFNL NLRP4 A*24 23 VYSSFVFNLF NLRP4 A*2424 KMLPEASLLI NLRP4 A*24 25 MLPEASLLI NLRP4 A*24 26 TYFFVDNQYW MMP12A*24 27 LSCTATPLF KHDC1L A*24 28 FWFDSREISF OR51E2 A*24 29 IYLLLPPVIOR51E2 A*24 30 RQAYSVYAF SLC45A3 A*24 31 KQMQEFFGL MMP1 A*24 32FYPEVELNF MMP1 A*24 33 FYQPDLKYLSF NLRP4 A*24 34 LIFALALAAF GAST A*24 35FSSTLVSLF MAGEC1 A*24 36 VYLASVAAF SLC45A3 A*24 37 ISFSDTVNVW ITIH6 A*2438 RYAHTLVTSVLF ITIH6 A*24 39 KTYLPTFETTI ENPP3, OR2A4 A*24 40NYPEGAAYEF ESR1 A*24 41 IYFATQVVF SLC45A3 A*24 42 VYDSIWCNM SCGB2A1 A*2443 KYKDHFTEI MAGEB1 A*24 44 FYHEDMPLW FCRL5 A*24 45 YGQSKPWTF PAX3 A*2446 IYPDSIQEL LOC645382, LOC645399, A*24 PRAMEF10 47 SYLWTDNLQEF DNAH17A*24 48 AWSPPATLFLF LOXL4 A*24 49 QYLSIAERAEF MSX1, MSX2 A*24 50RYFDENIQKF HEPHL1 A*24 51 YFDENIQKF HEPHL1 A*24 52 SWHKATFLF COL24A1A*24 53 LFQRVSSVSF HMCN1 A*24 54 SYQEAIQQL NEFH A*24 55 AVLRHLETF CDK6A*24 56 FYKLIQNGF FLT3 A*24 57 RYLQVVLLY NPSR1 A*24 58 IYYSHENLI F5 A*2459 VFPLVTPLL PTPRZ1 A*24 60 RYSPVKDAW KLHDC7B A*24 61 RIFTARLYF AICDAA*24 62 VYIVPVIVL OXTR A*24 63 LYIDKGQYL HMCN1 A*24 64 QFSHVPLNNF ALX1A*24 65 EYLLMIFKLV HRNR, RPTN A*24 66 IYKDYYRYNF PLA2G2D A*24 67SYVLQIVAI PTPRZ1 A*24 68 VYKEDLPQL EML5, EML6 A*24 69 KWFDSHIPRWERVV-1, ERVV-2 A*24 70 RYTGQWSEW IL9R A*24 71 RYLPNPSLNAF CYP1A1 A*24 72RWLDGSPVTL CLEC17A A*24 73 YFCSTKGQLF FCRL2 A*24 74 NYVLVPTMF CAPN6 A*2475 VYEHNHVSL BTBD16 A*24 76 IYIYPFAHW NPFFR2 A*24 77 LYGFFFKI BTBD16A*24 78 TYSKTIALYGF BTBD16 A*24 79 FYIVTRPLAF SUCNR1 A*24 80 SYATPVDLWCDK6 A*24 81 AYLKLLPMF SLC5A4 A*24 82 SYLENSASW DLX5 A*24 83 VLQGEFFLFKBTBD8 A*24 84 YTIERYFTL GABRP A*24 85 KYLSIPTVFF UGT1A3 A*24 86SYLPTAERL SYT12 A*24 87 NYTRLVLQF GABRP A*24 88 TYVPSTFLV GABRP A*24 89TYVPSTFLVVL GABRP A*24 90 TDLVQFLLF MAGEA10 A*24 91 KQQVVKFLILOC100288966, POTEB, A*24 POTEC, POTED, POTEE, POTEF, POTEG, POTEH,POTEI, POTEJ, POTEKP, POTEM 92 RALTETIMF ALPP, ALPPL2 A*24; B*15 93TDWSPPPVEF FAM178B A*24 94 THSGGTNLF MMP12 A*24; B*38 95 IGLSVVHRFOR51E2 A*24 96 SHIGVVLAF OR51E2 A*24; B*15 97 TQMFFIHAL OR51E2 A*24;B*39 98 LQIPVSPSF MAGEC1 A*24; B*15 99 ASAALTGFTF SLC45A3 A*24; A*32 100KVWSDVTPLTF MMP11 A*24; A*32 101 VYAVSSDRF DCX A*24 102 VLASAHILQFBTBD16 A*24 103 EMFFSPQVF ACTL8 A*24 104 GYGLTRVQPF ACTL8 A*24 105ITPATALLL LOC100996407, A*24 LOC101060778, LOC441242 106 LYAFLGSHFKISS1R A*24 107 FFFKAGFVWR MMP11 A*24 108 WFFQGAQYW MMP11 A*24 109AQHSLTQLF GPC2 A*24; B*15 110 VYSNPDLFW TRDV3 A*24 111 IRPDYSFQF TRDV3A*24 112 LYPDSVFGRLF SMC1B A*24 113 ALMSAFYTF MMP11 A*24 114 KALMSAFYTFMMP11 A*24 115 IMQGFIRAF PAEP A*24 116 TYFFVANKY MMP1 A*24 117RSMEHPGKLLF ESR1 A*24; B*15 118 IFLPFFIVF ADAM18 A*24 119 VWSCEGCKAFESR1, ESR2 A*24 120 VYAFMNENF QRFPR A*24 121 RRYFGEKVAL ANO7 A*24; B*27122 YFLRGRLYW MMP11 A*24 123 FFLQESPVF ABCC11 A*24 124 EYNVFPRTL MMP13A*24 125 LYYGSILYI LOC100996718, OR9G1, A*24 OR9G9 126 YSLLDPAQF SOX14A*24 127 FLPRAYYRW ANO7 A*24 128 AFQNVISSF NMUR2 A*24 129 IYVSLAHVL ANO7A*24 130 RPEKVFVF COL11A1 A*24 131 MHRTWRETF ANO7 A*24; A*32 132TFEGATVTL FCRL5 A*24 133 FFYVTETTF TERT A*24 134 IYSSQLPSF TFEC A*24 135KYKQHFPEI MAGEB17 A*24 136 YLKSVQLF RFX8 A*24 137 ALFAVCWAPF QRFPR A*24138 MMVTVVALF QRFPR A*24 139 AYAPRGSIYKF HHIPL2 A*24 140 IFQHFCEEI SMC1BA*24 141 QYAAAITNGL SALL3 A*24 142 PYWWNANMVF NOTUM A*24 143 KTKRWLWDFCOL11A1 A*24 144 LFDHGGTVFF ANO7 A*24 145 MYTIVTPML OR1N1 A*24 146NYFLDPVTI TRIM51, TRIM51HP A*24 147 FPYPSSILSV DLL3 A*24; B*51 148MLPQIPFLLL COL10A1 A*24; A*02 149 TQFFIPYTI COL10A1 A*24; A*32 150FIPVAWLIF MRGPRX4 A*24 151 RRLWAYVTI ITIH6 A*24 152 MHPGVLAAFLF MMP13A*24; B*15 153 AWSPPATLF LOXL4 A*24 154 DYSKQALSL LAMC2 A*24 155PYSIYPHGVTF F5 A*24 156 IYPHGVTFSP F5 A*24 157 SIYPHGVTF F5 A*24; B*15158 SYLKDPMIV DDX53 A*24 159 VFQPNPLF WISP3 A*24 160 YIANLISCF GLYATL3A*24 161 ILQAPLSVF FCRL5 A*24; B*15 162 YYIGIVEEY HEPHL1 A*24 163YYIGIVEEYW HEPHL1 A*24 164 MFQEMLQRL TRIML2 A*24 165 KDQPQVPCVF NAT1A*24; B*15 166 MMALWSLLHL ZACN A*24 167 LQPPWTTVF FCRL5 A*24; B*15 168LSSPVHLDF FCRL5 A*24; B*58 169 MYDLHHLYL EPYC A*24 170 IFIPATILL ACSM1A*24 171 LYTVPFNLI SLC45A2 A*24 172 RYFIAAEKILW HEPHL1 A*24 173RYLSVCERL NKX1-1, NKX1-2 A*24 174 TYGEEMPEEI DNAH17 A*24 175 SYFEYRRLLLAMC2 A*24 176 TQAGEYLLF FLT3 A*24 177 KYLITTFSL NLRP2 A*24 178AYPQIRCTW FLT3 A*24 179 MYNMVPFF DCT A*24 180 IYNKTKMAF SLCO6A1 A*24 181IHGIKFHYF NMUR2 A*24 182 AQGSGTVTF FCRL3 A*24; B*15 183 YQVAKGMEF FLT3A*24 184 VYVRPRVF HMCN1 A*24 185 LYICKVELM CTLA4 A*24 186 RRVTWNVLFBTBD17 A*24 187 KWFNVRMGFGF LIN28A, LIN28B A*24 188 SLPGSFIYVF HMCN1A*24 189 FYPDEDDFYF MYCN A*24 190 IYIIMQSCW FLT3 A*24 191 MSYSCGLPSLKRT33A A*24 192 CYSFIHLSF NLRP2, NLRP7 A*24 193 KYKPVALQCIA HMCN1 A*24194 EYFLPSLEII SLC24A5 A*24 195 IYNEHGIQQI COL11A1 A*24 196 VGRSPVFLFCOL11A1 A*24 197 YYHSGENLY PSG1, PSG3, PSG7 A*24 198 VLAPVSGQF FCRL5A*24; B*15 199 MFQFEHIKW FBXW10 A*24 200 LYMSVEDFI STK31 A*24 201VFPSVDVSF PTPRZ1 A*24 202 VYDTMIEKFA PTPRZ1 A*24 203 VYPSESTVM PTPRZ1A*24 204 WQNVTPLTF MMP16 A*24 205 ISWEVVHTVF HMCN1 A*24 206 EVVHTVFLFHMCN1 A*24; A*26 207 IYKFIMDRF FOXB1 A*24 208 QYLQQQAKL FOXB1 A*24 209DIYVTGGHLF KLHDC7B A*24 210 EAYSYPPATI HMCN1 A*24 211 MLYFAPDLIL PGRA*24 212 VYFVQYKIM IL22RA2 A*24 213 FYNRLTKLF OFCC1 A*24 214 YIPMSVMLFHTR7 A*24 215 KASKITFHW PTPRZ1 A*24 216 RHYHSIEVF LOXL4 A*24; C*12 217QRYGFSSVGF RHBG A*24; B*27 218 FYFYNCSSL ERVV-1, ERVV-2 A*24 219KVVSGFYYI CCR8 A*24 220 TYATHVTEI CCR8 A*24 221 VFYCLLFVF CCR8 A*24 222HYHAESFLF HEPHL1 A*24 223 KLRALSILF PTPRZ1 A*24 224 AYLQFLSVL GREB1 A*24225 ISMSATEFLL CYP1A1 A*24 226 TYSTNRTMI FLT3 A*24 227 YLPNPSLNAF CYP1A1A*24 228 VYLRIGGF WNT7A A*24 229 CAMPVAMEF KBTBD8 A*24 230 RWLSKPSLLKBTBD8 A*24 231 KYSVAFYSLD LAMA1 A*24 232 IWPGFTTSI PIWIL1 A*24 233LYSRRGVRTL DPPA3, DPPA3P2, A*24 LOC101060236 234 RYKMLIPF NLRP2 A*24 235VYISDVSVY CLECL1 A*24 236 LHLYCLNTF PGR A*24 237 RQGLTVLTW DNAH8 A*24238 YTCSRAVSLF OTOG A*24 239 IYTFSNVTF BTN1A1 A*24 240 RVHANPLLI APOBA*24; B*15 241 QKYYITGEAEGF ESR1 A*24 242 SYTPLLSYI C1orf94 A*24 243ALFPMGPLTF LILRA4 A*24; B*15 244 TYIDTRTVFL CAPN6 A*24 245 VLPLHFLPFHBG2 A*24 246 KIYTTVLFANI NPFFR2 A*24 247 VHSYLGSPF MPL A*24 248CWGPHCFEM SEMA5B A*24 249 HQYGGAYNRV DLX5 A*24 250 VYSDRQIYLL ABCC11A*24 251 DYLLSWLLF CNR2 A*24 252 RYLIIKYPF SUCNR1 A*24 253 QYYCLLLIFKLRF2 A*24 254 KQHAWLPLTI TCL1A A*24 255 VYLDEKQHAW TCL1A A*24 256QHAWLPLTI TCL1A A*24; B*39 257 MLILFFSTI OR56A3 A*24 258 VCWNPFNNTFRNF183 A*24 259 FFLFIPFF ADAM2 A*24 260 FLFIPFFIIF ADAM2 A*24 261IMFCLKNFWW TBC1D7 A*24 262 YIMFCLKNF TBC1D7 A*24 263 AYVTEFVSL SCN3AA*24 264 AYAIPSASLSW HMCN1 A*24 265 LYQQSDTWSL KIAA1407 A*24 266TQIITFESF CSF2 A*24; B*15 267 QHMLPFWTDL NLRP2 A*24 268 YQFGWSPNF CFHR5A*24 269 FSFSTSMNEF CAPN6 A*24 270 GTGKLFWVF BTLA A*24 271 INGDLVFSFCAPN6 A*24 272 IYFNHRCF SFMBT1 A*24 273 VTMYLPLLL GPR143 A*24 274EYSLPVLTF PTPRZ1 A*24 275 PEYSLPVLTF PTPRZ1 A*24 276 KFLGSKCSF HAS3 A*24277 MSAIWISAF SLC24A5 A*24 278 TYESVVTGFF HAS3 A*24 279 KYKNPYGF MMP20A*24 280 TIYSLEMKMSF GLB1L3 A*24 281 MDQNQVVWTF ROS1 A*24 282ASYQQSTSSFF FAM82A1 A*24 283 SYIVDGKII PSG9 A*24 284 QFYSTLPNTI ROS1A*24 285 YFLPGPHYF SOX30 A*24 286 HHTQLIFVF ELP4, EXOSC7, KCNG2, A*24TM4SF19, TOP2A 287 LVQPQAVLF PAX5 A*24 288 MGKGSISFLF PCSK1 A*24 289RTLNEIYHW FOXP3 A*24 290 VTPKMLISF OR5H8P A*24 291 YTRLVLQF GABRP A*24292 KMFPKDFRF TTLL6 A*24 293 MYAYAGWFY SLC7A11 A*24 294 KMGRIVDYF GABRPA*24 295 KYNRQSMTL APOB A*24 296 YQRPDLLLF GEN1 A*24; B*15 297 LKSPRLFTFBTBD16 A*24 298 TYETVMTFF BTBD16 A*24 299 FLPALYSLL CXCR3 A*24 300LFALPDFIF CXCR3 A*24 301 RTALSSTDTF CXCR3 A*24 302 YQGSLEVLF MROH2A A*24303 RFLDRGWGF ADAMTS12 A*24 304 YFGNPQKF LAMA3 A*24 305 RNAFSIYILMRGPRX1 A*24 306 RYILEPFFI SLC7A11 A*24 307 RILTEFELL TRIM31 A*24 308AAFISVPLLI TAS2R38 A*24 309 AFISVPLLI TAS2R38 A*24 310 EFINGWYVL MCOLN2A*24 311 IQNAILHLF OR51B5 A*24 312 YLCMLYALF KCNK18 A*24 313 IFMENAFELAPOB A*24 314 SQHFNLATF DNMT3B A*24; B*15 315 VYDYIPLLL MROH2A A*24 316IWAERIMF TDRD1 A*24 317 DWIWRILFLV IGHV1-58 A*24 318 VQADAKLLF FERMT1A*24; B*15 319 ATATLHLIF PCDHGB1, PCDHGB2 A*24; B*15 320 EVYQKIILKFPASD1 A*24 321 VYTVGHNLI KLB A*24 322 SFISPRYSWLF SPNS3 A*24 323NYSPVTGKF OTOL1 A*24 324 RYFVSNIYL PRSS21 A*24 325 IFMGAVPTL LPAR3 A*24326 VHMKDFFYF DYRK4 A*24 327 KWKPSPLLF GPR126 A*24 328 IYLVGGYSW KLHL14A*24 329 YLGKNWSF SPNS3 A*24 330 DYIQMIPEL RTL1 A*24 331 EYIDEFQSL RTL1A*24 332 VYCSLDKSQF RTL1 A*24 333 RYADLLIYTY MYO3B A*24 334 KVFGSFLTLAGTR2 A*24 335 RYQSVIYPF AGTR2 A*24 336 VYSDLHAFY MANEAL A*24; A*29 337SHSDHEFLF ARSH A*24; B*38 338 VYLTWLPGL IFNLR1 A*24 339 KQVIGIHTF SFMBT1A*24; B*15 340 FPPTPPLF BCL11A A*24 341 RYENVSILF ADCY8 A*24 342MYGIVIRTI NPSR1 A*24 343 EYQQYHPSL CLEC4C A*24 344 YAYATVLTF ABCC4 A*24;B*46 345 RYLEEHTEF MROH2A A*24 346 TYIDFVPYI TEX15 A*24 347 AWLIVLLFLCTCFL A*24 348 RSWENIPVTF C18orf54 A*24 349 IYMTTGVLL TDRD9 A*24 350VYKWTEEKF TSPEAR A*24 351 GYFGTASLF SLC16A14 A*24 352 NAFEAPLTF BRCA2A*24; C*07 353 AAFPGAFSF CRB2 A*24; C*07 354 QYIPTFHVY SLC44A5 A*24 355VYNNNSSRF MYO10 A*24 356 YSLEHLTQF ZCCHC16 A*24 357 RALLPSPLF SPATA31D1A*24 358 IYANVTEMLL CYP27C1 A*24 359 TQLPAPLRI GPR45 A*24 360 LYITKVTTIFSTL4 A*24 361 KQPANFIVL LOC100124692 A*24 362 NYMDTDNLMF LOC100124692A*24 363 QYGFNYNKF PNLDC1 A*24 364 KQSQVVFVL HMCN1, HMCN2, A*24;LOC101060175 B*48 365 KDLMKAYLF TXNDC16 A*24; B*37 366 RLGEFVLLF TGM6A*24 367 HWSHITHLF DPY19L1 A*24 368 AYFVAMHLF TENM4 A*24 369 NFYLFPTTFPNLDC1 A*24 370 TQMDVKLVF GEN1 A*24; B*15 371 FRSWAVQTF NOS2 A*24; C*06372 LYHNWRHAF PDE11A A*24 373 IWDALERTF ABCC11 A*24 374 MIFAVVVLF CCR4A*24 375 YYAADQWVF CCR4 A*24 376 KYVGEVFNI DMXL1 A*24 377 SLWREVVTFCEP250 A*24 378 VYAVISNIL TNR A*24 379 KLPTEWNVL AKAP13 A*24 380FYIRRLPMF CHRNA6 A*24 381 IYTDITYSF CHRNA6 A*24 382 SYPKELMKF MROH2AA*24

TABLE 1b Peptides according to the present invention. Seq ID HLA NoSequence Gene(s) allotype 463 VGGNVTSNF CT45A4, CT45A5 A*24 464VGGNVTSSF CT45A1, CT45A2, CT45A3, A*24 CT45A4, CT45A6, LOC101060208, LOC101060210, LOC101060211

TABLE 2 Additional peptides according to the presentinvention with no prior known cancer association. Seq ID No SequenceGene(s) HLA allotype 383 PYFSPSASF SPERT A*24 384 RTRGWVQTL C6orf223A*24 385 GYFGNPQKF LAMA3 A*24 386 YQSRDYYNF AR A*24 387 THAGVRLYF NUP155A*24; B*38

TABLE 3  Peptides according to the present invention usefulfor e.g. personalized cancer therapies. Seq ID HLA No Sequence Gene(s)allotype 388 LFHPEDTGQVF KLK3 A*24 389 AYSEKVTEF KLK2 A*24 390 KYKDYFPVILOC392555, MAGEC2 A*24 391 VYGEPRELL LOC392555, MAGEC2 A*24 392SYEKVINYL MAGEA9, MAGEA9B A*24 393 SYNDALLTF TRPM8 A*24 394 VYLPKIPSWKCNU1 A*24 395 NYEDHFPLL MAGEA10 A*24 396 SYVKVLHHLLOC101060230, MAGEA12 A*24 397 RMPTVLQCV KLK4 A*24 398 GYLQGLVSF KLK4A*24 399 VWSNVTPLKF MMP12 A*24 400 RYLEKFYGL MMP12 A*24 401 YLEKFYGLMMP12 A*24 402 TYKYVDINTF MMP12 A*24 403 LYFEKGEYF ACPP A*24 404SYLKAVFNL CYP4Z1, CYP4Z2P A*24 405 NYPKSIHSF MMP12 A*24 406 KYLEKYYNLMMP1 A*24 407 RILRFPWQL MMP11 A*24 408 VWSDVTPLTF MMP11 A*24 409VYTFLSSTL ESR1 A*24 410 IYISNSIYF CXorf48 A*24 411 VYPPYLNYL PGR A*24412 STIRGELFFF MMP11 A*24 413 RYMKKDYLI SLC35D3 A*24 414 TDSIHAWTFSLC35D3 A*24 415 KYEKIFEML CT45A1, CT45A2, A*24 CT45A3, CT45A4,CT45A5, CT45A6, LOC101060208, LOC101060210, LOC101060211 416 VFMKDGFFYFMMP1 A*24 417 GYIDKVRQL NEFH A*24 418 VHFEDTGKTLLF MMP13 A*24 419RYVFPLPYL SOX14 A*24 420 VYEKNGYIYF MMP13 A*24 421 RYILENHDF RFPL4B A*24422 VWSDVTPLNF MMP13 A*24 423 IYPDVTYAF CHRNA2 A*24 424 VQQWSVAVF PTHLHA*24 425 GYIDNVTLI LAMC2 A*24 426 SVHKITSTF LAMC2 A*24 427 VYFVAPAKFLAMC2 A*24 428 WYVNGVNYF TRPM8 A*24 429 YYSKSVGFMQW FAM111B A*24 430VYIAELEKI SMC1B A*24 431 KTPTNYYLF NMUR2 A*24 432 TRTGLFLRF NLRP2, NLRP7A*24 433 NYTSLLVTW PTPRZ1 A*24 434 VYDTMIEKF PTPRZ1 A*24 435 IYVTGGHLFKLHDC7B A*24 436 KYLQVVGMF OXTR A*24 437 VFKASKITF PTPRZ1 A*24 438SYSSCYSF KRT13, KRT17 A*24 439 VYALKVRTI CCR8 A*24 440 NYGVLHVTF NLRP11A*24 441 NYLVDPVTI TRIM43, TRIM43B A*24 442 KYLNSVQYI RALGPS2 A*24 443VFIHHLPQF ACSM1 A*24 444 IYLSDLTYI RALGPS2 A*24 445 TYIDTRTVF CAPN6 A*24446 IYGFFNENF NPFFR2 A*24 447 EYIRALQQL ASCL1 A*24 448 PFLPPAACFF ASCL1A*24 449 QYIEELQKF RALGPS2 A*24 450 QYDPTPLTW ADAMTS12 A*24 451FGLARIYSF CDK6 A*24 452 VYKDSIYYI KBTBD8 A*24 453 YYTVRNFTL PTPRZ1 A*24454 TLPNTIYRF ROS1 A*24 455 KYLSIPTVF UGT1A3 A*24 456 PYDPALGSPSRLF SP5A*24 457 LIFMLANVF GABRP A*24 458 DYLNEWGSRF CDH3 A*24 459 SYEVRSTFTAS2R38 A*24 460 VYPWLGALL CYP2W1 A*24

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as cancer, such as, for example, acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 387 and SEQ ID NO: 463 to SEQID: 464. More preferred are the peptides—alone or incombination—selected from the group consisting of SEQ ID NO: 1 to SEQ IDNO: 89 and SEQ ID NO: 463 to SEQ ID: 464 (see Table 1a and Table 1b),and their uses in the immunotherapy of acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer, and preferably acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.

Another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 387 and SEQ ID NO: 463 to SEQ ID: 464.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii) or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 387 and SEQ ID NO: 463 to SEQ ID:464, preferably containing SEQ ID No. 1 to SEQ ID No. 89 and SEQ ID NO:463 to SEQ ID: 464, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer, and preferably acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancercells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.The marker can be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length, but can be as short as8 amino acids in length, and as long as 10, 11, or 12 or longer, and incase of MHC class II peptides (elongated variants of the peptides of theinvention) they can be as long as 13, 14, 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it. A class I T cell“epitope” requires a short peptide that is bound to a class I MHCreceptor, forming a ternary complex (MHC class I alpha chain,beta-2-microglobulin, and peptide) that can be recognized by a T cellbearing a matching T-cell receptor binding to the MHC/peptide complexwith appropriate affinity. Peptides binding to MHC class I molecules aretypically 8-14 amino acids in length, and most typically 9 amino acidsin length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 4 Expression frequencies F of HLA-A*02, HLA-A*01, HLA-A*03,HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 serotypes. Haplotypefrequencies Gf are derived from a study which used HLA-typing data froma registry of more than 6.5 million volunteer donors in the U.S.(Gragert et al., 2013). The haplotype frequency is the frequency of adistinct allele on an individual chromosome. Due to the diploid set ofchromosomes within mammalian cells, the frequency of genotypicoccurrence of this allele is higher and can be calculated employing theHardy-Weinberg principle (F = 1 − (1-Gf)²). Calculated phenotype AllelePopulation from allele frequency (F) A*02 African (N = 28557) 32.3%European Caucasian (N = 1242890) 49.3% Japanese (N = 24582) 42.7%Hispanic, S + Cent Amer. 46.1% (N = 146714) Southeast Asian (N = 27978)30.4% A*01 African (N = 28557) 10.2% European Caucasian (N = 1242890)30.2% Japanese (N = 24582)  1.8% Hispanic, S + Cent Amer. 14.0% (N =146714) Southeast Asian (N = 27978) 21.0% A*03 African (N = 28557) 14.8%European Caucasian (N = 1242890) 26.4% Japanese (N = 24582)  1.8%Hispanic, S + Cent Amer. 14.4% (N = 146714) Southeast Asian (N = 27978)10.6% A*24 African (N = 28557)  2.0% European Caucasian (N = 1242890) 8.6% Japanese (N = 24582) 35.5% Hispanic, S + Cent Amer. 13.6% (N =146714) Southeast Asian (N = 27978) 16.9% B*07 African (N = 28557) 14.7%European Caucasian (N = 1242890) 25.0% Japanese (N = 24582) 11.4%Hispanic, S + Cent Amer. 12.2% (N = 146714) Southeast Asian (N = 27978)10.4% B*08 African (N = 28557)  6.0% European Caucasian (N = 1242890)21.6% Japanese (N = 24582)  1.0% Hispanic, S + Cent Amer.  7.6% (N =146714) Southeast Asian (N = 27978)  6.2% B*44 African (N = 28557) 10.6%European Caucasian (N = 1242890) 26.9% Japanese (N = 24582) 13.0%Hispanic, S + Cent Amer. 18.2% (N = 146714) Southeast Asian (N = 27978)13.1%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*24. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that areA*24-positive, whereas no selection for MHC class II allotypes isnecessary due to the pan-binding nature of these peptides.

If A*24 peptides of the invention are combined with peptides binding toanother allele, for example A*02, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom allelefrequencies.net).

TABLE 5 HLA alleles coverage in European Caucasian population(calculated from (Gragert et al., 2013)). coverage (at combined leastone A- combined combined with B*07 allele) with B*07 with B*44 and B*44A*02/A*01 70% 78% 78% 84% A*02/A*03 68% 76% 76% 83% A*02/A*24 61% 71%71% 80% A*′01/A*03 52% 64% 65% 75% A*01/A*24 44% 58% 59% 71% A*03/A*2440% 55% 56% 69% A*02/A*01/A*03 84% 88% 88% 91% A*02/A*01/A*24 79% 84%84% 89% A*02/A*03/A*24 77% 82% 83% 88% A*01/A*03/A*24 63% 72% 73% 81%A*02/A*01/ 90% 92% 93% 95% A*03/A*24

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:percent identity=100[1−(C/R)]wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences;and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 387 and SEQ ID NO: 463 to SEQ ID NO: 464 or avariant thereof which is 88% homologous to SEQ ID NO: 1 to SEQ ID NO:387 and SEQ ID NO: 463 to SEQ ID NO: 464, or a variant thereof that willinduce T cells cross-reacting with said peptide. The peptides of theinvention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” shall refer to thedegree of identity (see percent identity above) between sequences of twoamino acid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 387 and SEQ IDNO: 463 to SEQ ID NO: 464. For example, a peptide may be modified sothat it at least maintains, if not improves, the ability to interactwith and bind to the binding groove of a suitable MHC molecule, such asHLA-A*02 or -DR, and in that way it at least maintains, if not improves,the ability to bind to the TCR of activated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Godkin etal., 1997; Rammensee et al., 1999), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 387 and SEQ ID NO: 463 to SEQ ID NO: 464, by maintainingthe known anchor residues, and would be able to determine whether suchvariants maintain the ability to bind MHC class I or II molecules. Thevariants of the present invention retain the ability to bind to the TCRof activated T cells, which can subsequently cross-react with and killcells that express a polypeptide containing the natural amino acidsequence of the cognate peptide as defined in the aspects of theinvention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or—II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or —II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motives of the peptides according to SEQ ID NO: 7,10, 23, and 40 Position 1 2 3 4 5 6 7 8 9 SEQ ID No 7 G Y P L R G S S IVariant L F F F L F F Position 1 2 3 4 5 6 7 8 9 SEQ ID No 10 Q Y P E FS I E L Variant I F F I F F F Position 1 2 3 4 5 6 7 8 9 10 SEQ ID No 23V Y S S F V F N L F Variant I L F I F L F Position 1 2 3 4 5 6 7 8 9 10SEQ ID No 40 N Y P E G A A Y E F Variant I L F I F L F

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to a MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 387 and SEQ ID NO: 463 to SEQ ID NO: 464.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any one ofSEQ ID NO: 1 to SEQ ID NO 387 and SEQ ID NO: 463 to SEQ ID NO: 464 or avariant thereof contains additional N- and/or C-terminally locatedstretches of amino acids that are not necessarily forming part of thepeptide that functions as an epitope for MHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004 (Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich (sigma-aldrich.com)provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 387and SEQ ID NO: 463 to SEQ ID NO: 464 and has been synthetically produced(e.g. synthesized) as a pharmaceutically acceptable salt. Methods tosynthetically produce peptides are well known in the art. The salts ofthe peptides according to the present invention differ substantiallyfrom the peptides in their state(s) in vivo, as the peptides asgenerated in vivo are no salts. The non-natural salt form of the peptidemediates the solubility of the peptide, in particular in the context ofpharmaceutical compositions comprising the peptides, e.g. the peptidevaccines as disclosed herein. A sufficient and at least substantialsolubility of the peptide(s) is required in order to efficiently providethe peptides to the subject to be treated. Preferably, the salts arepharmaceutically acceptable salts of the peptides. These salts accordingto the invention include alkaline and earth alkaline salts such as saltsof the Hofmeister series comprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻,Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻, I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺,Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts areselected from (NH₄)₃PO₄, (NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO,NH₄Cl, NH₄Br, NH₄NO₃, NH₄ClO₄, NH₄I, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄,Rb₂SO₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄I, Rb₄SCN, K₃PO₄,K₂HPO₄, KH₂PO₄, K₂SO₄, KCH₃COO, KCl, KBr, KNOB, KClO₄, KI, KSCN, Na₃PO₄,Na₂HPO₄, NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI,NaSCN, ZnCl₂ Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr,CsNO₃, CsClO₄, CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO,LiCl, LiBr, LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄,Mg(H₂PO₄)₂, Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂,MgI₂, Mg(SCN)₂, MnCl₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄,Ca(CH₃COO)₂, CaCl₂), CaBr₂, Ca(NO₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂,Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂, BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂,Ba(NO₃)₂, Ba(ClO₄)₂, BaI₂, and Ba(SCN)₂. Particularly preferred are NHacetate, MgCl₂, KH₂PO₄, Na₂SO₄, KCI, NaCl, and CaCl₂), such as, forexample, the chloride or acetate (trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981) and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by any one, or a combination of,techniques such as re-crystallization, size exclusion chromatography,ion-exchange chromatography, hydrophobic interaction chromatography and(usually) reverse-phase high performance liquid chromatography usinge.g. acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A-1J).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated, and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from acute myeloid leukemia,breast cancer, cholangiocellular carcinoma, chronic lymphocyticleukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer samples (N=263samples) with the fragmentation patterns of corresponding syntheticreference peptides of identical sequences. Since the peptides weredirectly identified as ligands of HLA molecules of primary tumors, theseresults provide direct evidence for the natural processing andpresentation of the identified peptides on primary cancer tissueobtained from 263 acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma, anduterine and endometrial cancer patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (see example1). All TUMAPs contained in the present application were identified withthis approach on acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer samples confirming their presentation onacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.

TUMAPs identified on multiple acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer and normal tissues were quantified usingion-counting of label-free LC-MS data. The method assumes that LC-MSsignal areas of a peptide correlate with its abundance in the sample.All quantitative signals of a peptide in various LC-MS experiments werenormalized based on central tendency, averaged per sample and mergedinto a bar plot, called presentation profile. The presentation profileconsolidates different analysis methods like protein database search,spectral clustering, charge state deconvolution (decharging) andretention time alignment and normalization.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 2A-2Q).Peptides which are derived from proteins whose coding mRNA is highlyexpressed in cancer tissue, but very low or absent in vital normaltissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancersamples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy blood cells, blood vessels, brain, heart, liver, lung, adiposetissue, adrenal gland, bile duct, bladder, bone marrow, esophagus, eye,gallbladder, head & neck, large intestine, small intestine, kidney,lymph node, peripheral nerve, pancreas, parathyroid gland, peritoneum,pituitary, pleura, skeletal muscle, skin, spleen, stomach, thyroid,trachea, ureter cells or other normal tissue cells, demonstrating a highdegree of tumor association of the source genes (see Example 2).Moreover, the peptides themselves are strongly over-presented on tumortissue—“tumor tissue” in relation to this invention shall mean a samplefrom a patient suffering from acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer, but not on normal tissues (see Example1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill, and can be found in the respectiveliterature as well (see also below). Thus, the peptides of the presentinvention are useful for generating an immune response in a patient bywhich tumor cells can be destroyed. An immune response in a patient canbe induced by direct administration of the described peptides orsuitable precursor substances (e.g. elongated peptides, proteins, ornucleic acids encoding these peptides) to the patient, ideally incombination with an agent enhancing the immunogenicity (i.e. anadjuvant). The immune response originating from such a therapeuticvaccination can be expected to be highly specific against tumor cellsbecause the target peptides of the present invention are not presentedon normal tissues in comparable copy numbers, preventing the risk ofundesired autoimmune reactions against normal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides according to the invention capable of binding toTCRs and antibodies when presented by an MHC molecule.

The present description also relates to fragments of the TCRs accordingto the invention that are capable of binding to a peptide antigenaccording to the present invention when presented by an HLA molecule.The term particularly relates to soluble TCR fragments, for example TCRsmissing the transmembrane parts and/or constant regions, single chainTCRs, and fusions thereof to, for example, with lg.

The present description also relates to nucleic acids, vectors and hostcells for expressing TCRs and peptides of the present description; andmethods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V) and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise, the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A-restricted tumor-associated self-antigens. It is now known,although tumor antigens have the potential to be immunogenic, becausetumors arise from the individual's own cells only mutated proteins orproteins with altered translational processing will be seen as foreignby the immune system. Antigens that are upregulated or overexpressed (socalled self-antigens) will not necessarily induce a functional immuneresponse against the tumor: T-cells expressing TCRs that are highlyreactive to these antigens will have been negatively selected within thethymus in a process known as central tolerance, meaning that onlyT-cells with low-affinity TCRs for self-antigens remain. Therefore,affinity of TCRs or variants of the present description to pepides canbe enhanced by methods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*024-negative healthy donors withA24/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluo-rescence activatedcell sorting (FACS)—Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)—Calibur analysis.

In one aspect, in order to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs(Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors, or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH₂ group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluene sulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pureor combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by CD4T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 387 and SEQ IDNo. 463 to SEQ ID No. 464, and at least one additional peptide,preferably two to 50, more preferably two to 25, even more preferablytwo to 20 and most preferably two, three, four, five, six, seven, eight,nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen or eighteen peptides. The peptide(s) may be derived from oneor more specific TAAs and may bind to MHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbás and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. Regarding vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantially pureor contained in a suitable vector or delivery system. The nucleic acidmay be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, bevacizumab, interferon-alpha, CpGoligonucleotides and derivates, poly-(I:C) and derivates, RNA,sildenafil, and particulate formulations with PLG or virosomes.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any one of SEQ ID NO 1 to SEQ ID NO 387 andSEQ ID No. 463 to SEQ ID No. 464, according to the invention at handwith the MHC molecule, using the cell-SELEX (Systematic Evolution ofLigands by Exponential enrichment) technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen (preferably a peptide according to thepresent invention), the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically binding to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen.

It is thus a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with an HLA-restricted antigen, whereinthe antibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 387 andSEQ ID No. 463 to SEQ ID No. 464, or a variant thereof which is at least88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ ID NO: 387and SEQ ID No. 463 to SEQ ID No. 464 or a variant thereof that induces Tcells cross-reacting with said peptide, wherein said peptide is not theunderlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:387 and SEQ ID No. 463 to SEQ ID No. 464 or a variant thereof which isat least 88% homologous (preferably identical) to SEQ ID NO: 1 to SEQ IDNO: 387 and SEQ ID No. 463 to SEQ ID No. 464, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 387 and SEQID No. 463 to SEQ ID No. 464.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of acute myeloid leukemia,breast cancer, cholangiocellular carcinoma, chronic lymphocyticleukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 387 and SEQ ID No. 463 to SEQ ID No. 464 or said variant aminoacid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as according to the present invention.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer cells or other solidor hematological tumor cells such as acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lungcancer (including non-small cell lung cancer adenocarcinoma, squamouscell non-small cell lung cancer, and small cell lung cancer), ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, urinary bladder carcinoma, uterine and endometrialcancer. The present invention also relates to the use of these noveltargets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of an acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer marker (poly)peptide,delivery of a toxin to an acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer cell expressing a cancer marker gene atan increased level, and/or inhibiting the activity of an acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancermarker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer marker polypeptides or fragments thereofmay be used to generate the antibodies of the invention. A polypeptideto be used for generating an antibody of the invention may be partiallyor fully purified from a natural source or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 387and SEQ ID No. 463 to SEQ ID No. 464 polypeptide, or a variant orfragment thereof, can be expressed in prokaryotic cells (e.g., bacteria)or eukaryotic cells (e.g., yeast, insect, or mammalian cells), afterwhich the recombinant protein can be purified and used to generate amonoclonal or polyclonal antibody preparation that specifically bind theacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancermarker polypeptide used to generate the antibody according to theinvention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used, and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer,the efficacy of the therapeutic antibody can be assessed in various wayswell known to the skilled practitioner. For instance, the size, number,and/or distribution of cancer in a subject receiving treatment may bemonitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography. In one embodiment, antibodies orfragments thereof bind to the extracellular domains of two or moretargets of a protein selected from the group consisting of theabove-mentioned proteins, and the affinity value (Kd) is less than 1×10μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of an MHC class I epitope being used as an antigen; the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 387 and SEQ ID No. 463to SEQ ID No. 464, or a variant amino acid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e.g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 387and SEQ ID No. 463 to SEQ ID No. 464.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut in the tumor it is expressed. By “over-expressed” the inventors meanthat the polypeptide is present at a level at least 1.2-fold of thatpresent in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;

(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and

(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed, or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer,the medicament of the invention is preferably used to treat acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancerpatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from several acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancertissues, the warehouse may contain HLA-A*02, HLA-A*01, HLA-A*03,HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 marker peptides. Thesepeptides allow comparison of the magnitude of T-cell immunity induced byTUMAPS in a quantitative manner and hence allow important conclusion tobe drawn on the capacity of the vaccine to elicit anti-tumor responses.Secondly, they function as important positive control peptides derivedfrom a “non-self” antigen in the case that any vaccine-induced T-cellresponses to TUMAPs derived from “self” antigens in a patient are notobserved. And thirdly, it may allow conclusions to be drawn, regardingthe status of immunocompetence of the patient.

TUMAPs for the warehouse are identified by using an integratedfunctional genomics approach combining gene expression analysis, massspectrometry, and T-cell immunology (XPresident®). The approach assuresthat only TUMAPs truly present on a high percentage of tumors but not oronly minimally expressed on normal tissue, are chosen for furtheranalysis. For initial peptide selection, acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer samples from patientsand blood from healthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry

2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer)compared with a range of normal organs and tissues3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancerpatients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patients' tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide-based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer cells and since it was determined thatthese peptides are not or at lower levels present in normal tissues,these peptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lungcancer (including non-small cell lung cancer adenocarcinoma, squamouscell non-small cell lung cancer, and small cell lung cancer), ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, urinary bladder carcinoma, uterine and endometrialcancer. Presence of groups of peptides can enable classification orsub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance.

Thus, presence of peptides shows that this mechanism is not exploited bythe analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

FIGURES

FIGS. 1A through 1J show the over-presentation of various peptides indifferent cancer tissues (black dots). Upper part: Median MS signalintensities from technical replicate measurements are plotted as dotsfor single HLA-A*24 positive normal (grey dots, left part of figure) andtumor samples (black dots, right part of figure) on which the peptidewas detected. Boxes display median, 25th and 75th percentile ofnormalized signal intensities, while whiskers extend to the lowest datapoint still within 1.5 interquartile range (IQR) of the lower quartile,and the highest data point still within 1.5 IQR of the upper quartile.Normal organs are ordered according to risk categories (blood cells,blood vessels, brain, liver, lung: high risk, grey dots; reproductiveorgans, breast, prostate: low risk, grey dots; all other organs: mediumrisk; grey dots). Lower part: The relative peptide detection frequencyin every organ is shown as spine plot. Numbers below the panel indicatenumber of samples on which the peptide was detected out of the totalnumber of samples analyzed for each organ (N=75 for normal samples,N=263 for tumor samples). If the peptide has been detected on a samplebut could not be quantified for technical reasons, the sample isincluded in this representation of detection frequency, but no dot isshown in the upper part of the figure. Tissues (from left to right):Normal samples: blood cells; bloodvess (blood vessels); brain; heart;liver; lung; adrenal gl (adrenal gland); bile duct; gall bl(gallbladder); intest. la (large intestine); intest. sm (smallintestine); kidney; nerve perith (peripheral nerve); pancreas; pituit(pituitary); skin; spinal cord; spleen; stomach; thyroid. Tumor samples:AML (acute myeloid leukemia); BRCA (breast cancer); CCC(cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC(colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC(gastric cancer); HCC (hepatocellular carcinoma); HNSCC (head and necksquamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma);NSCLCadeno (non-small cell lung cancer adenocarcinoma); NSCLCother(NSCLC samples that could not unambiguously be assigned to NSCLCadeno orNSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC(ovarian cancer); OSCAR (esophageal cancer); PACA (pancreatic cancer);PRCA (prostate cancer); RCC (renal cell carcinoma); SCLC (small celllung cancer); UBC (urinary bladder carcinoma); UEC (uterine andendometrial cancer). FIG. 1A) Gene symbol: SLC6A3, Peptide: FMVIAGMPLF(SEQ ID NO.: 15), FIG. 1B) Gene symbol: KLHDC7B, Peptide: RYSPVKDAW (SEQID NO.: 60), FIG. 1C) Gene symbol: CAPN6, Peptide: NYVLVPTMF (SEQ IDNO.: 74), FIG. 1D) Gene symbol: SYT12, Peptide: SYLPTAERL (SEQ ID NO.:86), FIG. 1E) Gene symbol: PTPRZ1, Peptide: VYDTMIEKFA (SEQ ID NO.:202), FIG. 1F) Gene symbol: PTPRZ1, Peptide: EYSLPVLTF (SEQ ID NO.:274), FIG. 1G) Gene symbol: LOC100124692, Peptide: NYMDTDNLMF (SEQ IDNO.: 362), FIG. 1H) Gene symbol: AR, Peptide: YQSRDYYNF (SEQ ID NO.:386), FIG. 1I) Gene symbol: CT45A4, CT45A5, Peptide: VGGNVTSNF (SEQ IDNO.: 463), and FIG. 1J) Gene symbol: CT45A1, CT45A2, CT45A3, CT45A4,CT45A6, LOC101060208, LOC101060210, LOC101060211, Peptide: VGGNVTSSF(SEQ ID NO.: 464).

FIGS. 2A through 2Q show exemplary expression profile of source genes ofthe present invention that are over-expressed in different cancersamples. Tumor (black dots) and normal (grey dots) samples are groupedaccording to organ of origin. Box-and-whisker plots represent medianFPKM value, 25th and 75th percentile (box) plus whiskers that extend tothe lowest data point still within 1.5 interquartile range (IQR) of thelower quartile and the highest data point still within 1.5 IQR of theupper quartile. Normal organs are ordered according to risk categories.FPKM: fragments per kilobase per million mapped reads. Tissues (fromleft to right): Normal samples: blood cells; bloodvess (blood vessels);brain; heart; liver; lung; adipose (adipose tissue); adrenal gl (adrenalgland); bile duct; bladder; bone marrow; esoph (esophagus); eye; gall bl(gallbladder); head&neck; intest. la (large intestine); intest. sm(small intestine); kidney; lymph node; nerve perith (peripheral nerve);pancreas; parathyr (parathyroid gland); perit (peritoneum); pituit(pituitary); pleura; skel. mus (skeletal muscle); skin; spleen; stomach;thyroid; trachea; ureter; breast; ovary; placenta; prostate; testis;thymus; uterus. Tumor samples: AML (acute myeloid leukemia); BRCA(breast cancer); CCC (cholangiocellular carcinoma); CLL (chroniclymphocytic leukemia); CRC (colorectal cancer); GBC (gallbladdercancer); GBM (glioblastoma); GC (gastric cancer); HCC (hepatocellularcarcinoma); HNSCC (head and neck squamous cell carcinoma); MEL(melanoma); NHL (non-Hodgkin lymphoma); NSCLCadeno (non-small cell lungcancer adenocarcinoma); NSCLCother (NSCLC samples that could notunambiguously be assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam(squamous cell non-small cell lung cancer); OC (ovarian cancer); OSCAR(esophageal cancer); PACA (pancreatic cancer); PRCA (prostate cancer);RCC (renal cell carcinoma); SCLC (small cell lung cancer); UBC (urinarybladder carcinoma); UEC (uterine and endometrial cancer). FIG. 2A) Genesymbol: MAGEA4, Peptide: IFPKTGLLII (SEQ ID No.: 1), FIG. 2B) Genesymbol: TRPM8, Peptide: KFLTHDVLTELF (SEQ ID No.: 3), FIG. 2C) Genesymbol: CHRNA9, Peptide: KYYIATMAL (SEQ ID No.: 13), FIG. 2D) Genesymbol: MMP12, Peptide: KYVDINTFRL (SEQ ID No.: 18), FIG. 2E) Genesymbol: SPINK2, Peptide: LYMRFVNTHF (SEQ ID No.: 21), FIG. 2F) Genesymbol: OR51E2, Peptide: FWFDSREISF (SEQ ID No.: 28), FIG. 2G) Genesymbol: MMP1, Peptide: KQMQEFFGL (SEQ ID No.: 31), FIG. 2H) Gene symbol:MAGEC1, Peptide: FSSTLVSLF (SEQ ID No.: 35), FIG. 2I) Gene symbol:ENPP3, Peptide: KTYLPTFETTI (SEQ ID No.: 39), FIG. 2J) Gene symbol:POTEG, POTEH, Peptide: MVLQPQPQLF (SEQ ID No.: 4), FIG. 2K) Gene symbol:OR51E2, Peptide: TQMFFIHAL (SEQ ID No.: 97), FIG. 2L) Gene symbol:MMP11, Peptide: FFFKAGFVWR (SEQ ID No.: 107), FIG. 2M) Gene symbol:MMP11, Peptide: YFLRGRLYW (SEQ ID No.: 122), FIG. 2N) Gene symbol:SLC24A5, Peptide: EYFLPSLEII (SEQ ID No.: 194), FIG. 2O) Gene symbol:SLC24A5, Peptide: MSAIWISAF (SEQ ID No.: 277), FIG. 2P) Gene symbol:ELP4, EXOSC7, KCNG2, TM4SF19, TOP2A, Peptide: HHTQLIFVF (SEQ ID No.:286), FIG. 2Q) Gene symbol: LAMA3, Peptide: YFGNPQKF (SEQ ID No.: 304).

FIGS. 3A through 3G show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*24+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*24 incomplex with SEQ ID NO: 420 peptide (VYEKNGYIYF, Seq ID NO: 420) (A,left panel), SEQ ID NO: 411 peptide (VYPPYLNYL, Seq ID NO: 411) (B, leftpanel), SEQ ID NO: 5 peptide (LQPQPQLFFSF, Seq ID NO: 5) (C, leftpanel), SEQ ID NO: 77 peptide (LYGFFFKI, Seq ID NO: 77) (D, left panel),SEQ ID NO: 76 peptide (IYIYPFAHW, Seq ID NO: 76) (E, left panel), SEQ IDNO: 32 peptide (FYPEVELNF, Seq ID NO: 32) (F, left panel), SEQ ID NO: 23peptide (VYSSFVFNLF, Seq ID NO: 23) (G, left panel), respectively. Afterthree cycles of stimulation, the detection of peptide-reactive cells wasperformed by 2D multimer staining with A*24/SEQ ID NO: 420 (A), A*24/SEQID NO: 411 (B), A*24/SEQ ID NO: 5 (C), A*24/SEQ ID NO: 77 (D), A*24/SEQID NO: 76 (E), A*24/SEQ ID NO: 32 (F), A*24/SEQ ID NO: 23 (G),respectively. Right panels (A) show control staining of cells stimulatedwith irrelevant A*24/peptide complexes. Viable singlet cells were gatedfor CD8+ lymphocytes. Boolean gates helped excluding false-positiveevents detected with multimers specific for different peptides.Frequencies of specific multimer+ cells among CD8+ lymphocytes areindicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface Tissue Samples

Patients' tumor tissues were obtained from: Asterand Bioscience (DetroitUSA & Royston, Herts, UK), BioServe (Beltsville, Md., USA), ConversantBio (Huntsville, Ala., USA), Geneticist Inc. (Glendale, Calif., USA),Heidelberg University Hospital (dept. of Neurosurgery, Heidelberg,Germany), Heidelberg University Hospital (General, Visceral andTransplantation Surgery, Heidelberg, Germany), Heidelberg UniversityHospital (Thoraxklinik, Heidelberg, Germany), Istituto Nazionale Tumori“Pascale” (Molecular Biology and Viral Oncology Unit, Naples, Italy),Kyoto Prefectural University of Medicine (Kyoto, Japan), LeidenUniversity Medical Center (LUMC) (Leiden, Netherlands), Osaka CityUniversity (Osaka, Japan), ProteoGenex Inc. (Culver City, Calif., USA),Saint Savvas Hospital (Athens, Greece), Tissue Solutions (Glasgow, UK),University Hospital Bonn (dept. of Internal Medicine III, Hematology andOncology, Bonn, Germany), University Hospital Tübingen (Dept. ofGeneral, Visceral and Transplant Surgery, Tübingen, Germany), UniversityHospital Tübingen (Dept. of Immunology, Tübingen, Germany), UniversityHospital Tübingen (Dept. of Urology, Tübingen, Germany), University ofGeneva (Division of Oncology, Geneva, Switzerland)

Normal tissues were obtained from: Asterand Bioscience (Detroit USA &Royston, Herts, UK), BioServe (Beltsville, Md., USA), Capital BioScienceInc. (Rockville, Md., USA), Centre for Clinical Transfusion MedicineTuebingen (Tübingen, Germany), Geneticist Inc. (Glendale, Calif., USA),Heidelberg University Hospital (Thoraxklinik, Heidelberg, Germany),Kyoto Prefectural University of Medicine (Kyoto, Japan), ProteoGenexInc. (Culver City, Calif., USA), University Hospital Tübingen (Dept. ofGeneral, Visceral and Transplant Surgery, Tübingen, Germany), UniversityHospital Tübingen (Dept. of Urology, Tübingen, Germany)

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, the HLA-DR specific antibody L243 and the HLA DP specificantibody B7/21, CNBr-activated sepharose, acid treatment, andultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST at a fixed false discovery rate (q≤0.05) and additional manualcontrol. In cases where the identified peptide sequence was uncertain itwas additionally validated by comparison of the generated naturalpeptide fragmentation pattern with the fragmentation pattern of asynthetic sequence-identical reference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus each identified peptidecan be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer samples to a baseline of normal tissuesamples. Presentation profiles of exemplary over-presented peptides areshown in FIGS. 1A-1J.

Table 8a and Table 8b show the presentation on various cancer entitiesfor selected peptides, and thus the particular relevance of the peptidesas mentioned for the diagnosis and/or treatment of the cancers asindicated (e.g. peptide SEQ ID No. 3 for prostate cancer (PRCA), peptideSEQ ID No. 20 for ovarian cancer (OC) and pancreatic cancer (PACA)).

TABLE 8a Overview of presentation of selected tumor-associated peptides of the present invention across entities. SEQ IDPeptide Presentation on No. Sequence cancer entities 1 IFPKTGLLIINSCLCadeno, NSCLCsquam, OSCAR 2 LYAPTILLW CRC, GC, NSCLCadeno,NSCLCsquam, UEC 3 KFLTHDVLTELF PRCA 4 MVLQPQPQLF NSCLCadeno 5LQPQPQLFFSF PRCA 6 IVTFMNKTLGTF NSCLCadeno 7 GYPLRGSSI GC 9 TYINSLAILNSCLCadeno, PRCA, RCC, UBC 10 QYPEFSIEL PRCA 11 RAMCAMMSF PACA 12KYMSRVLFVY CCC 13 KYYIATMAL GC 14 YYIATMALI PRCA 15 FMVIAGMPLFAML, CLL, GC, HCC, NHL, NSCLCother 16 GYFLAQYLM RCC 17 IYPEAIATLBRCA, NSCLCadeno, NSCLCother, RCC 18 KYVDINTFRL NSCLCsquam 19 ILLCMSLLLFNSCLCadeno, NSCLCsquam, PRCA 20 ELMAHPFLL OC, PACA 21 LYMRFVNTHF AML 22VYSSFVFNL NHL 23 VYSSFVFNLF NHL 24 KMLPEASLLI NHL 25 MLPEASLLI NHL 26TYFFVDNQYW NSCLCsquam, OSCAR 27 LSCTATPLF NSCLCadeno 28 FWFDSREISF PRCA29 IYLLLPPVI NSCLCadeno, PRCA 30 RQAYSVYAF PRCA 31 KQMQEFFGLGC, NSCLCsquam 32 FYPEVELNF CCC, UBC 33 FYQPDLKYLSF NHL 34 LIFALALAAFUEC 35 FSSTLVSLF OC 36 VYLASVAAF PRCA 37 ISFSDTVNVW RCC 38 RYAHTLVTSVLFBRCA, MEL 39 KTYLPTFETTI UEC 40 NYPEGAAYEF NSCLCadeno, OC 41 IYFATQVVFPRCA 42 VYDSIWCNM UEC 43 KYKDHFTEI BRCA, GBC, NHL, NSCLCadeno,NSCLCsquam 44 FYHEDMPLW NHL 45 YGQSKPWTF HCC, HNSCC, NSCLCadeno,NSCLCsquam, OSCAR, RCC 46 IYPDSIQEL HCC 47 SYLWTDNLQEF OSCAR 48AWSPPATLFLF GC, MEL, NSCLCsquam, OC, RCC 49 QYLSIAERAEF GC, UBC 50RYFDENIQKF OSCAR 51 YFDENIQKF NSCLCsquam, OSCAR 52 SWHKATFLF AML 53LFQRVSSVSF HCC 54 SYQEAIQQL PRCA 55 AVLRHLETF CCC 56 FYKLIQNGF AML 57RYLQVVLLY GC 58 IYYSHENLI CCC, CRC, GC, HCC, NSCLCadeno, NSCLCsquam, OC,PRCA 59 VFPLVTPLL GBM 60 RYSPVKDAW CCC, GBC, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR 61 RIFTARLYF NHL 62 VYIVPVIVLGBM, NSCLCsquam 63 LYIDKGQYL GBM, HNSCC, MEL, NSCLCadeno, NSCLCsquam 64QFSHVPLNNF NSCLCsquam 65 EYLLMIFKLV NSCLCadeno, NSCLCsquam, PRCA 66IYKDYYRYNF NHL 67 SYVLQIVAI GBM, MEL 68 VYKEDLPQL CLL, NSCLCsquam 69KWFDSHIPRW GBC 70 RYTGQWSEW UBC 71 RYLPNPSLNAF HCC, NSCLCadeno 72RWLDGSPVTL NHL 73 YFCSTKGQLF CLL, NHL 74 NYVLVPTMFCCC, CRC, GBC, GC, HCC, NSCLCadeno, NSCLCsquam, OC, UEC 75 VYEHNHVSL UBC76 IYIYPFAHW HCC, NHL 77 LYGFFFKI GC, HCC, UBC 78 TYSKTIALYGF UBC 79FYIVTRPLAF NHL, NSCLCadeno 80 SYATPVDLW AML, CCC, MEL, NSCLCsquam 81AYLKLLPMF BRCA, HNSCC, MEL 82 SYLENSASW BRCA, HNSCC, NSCLCadeno,OSCAR, SCLC, UEC 83 VLQGEFFLF NHL 84 YTIERYFTL GC, NSCLCsquam, UEC 85KYLSIPTVFF CCC, GBC, GC, HCC, NSCLCadeno, PRCA, RCC, UBC 86 SYLPTAERLGBC, GBM, GC, HCC, NSCLCadeno, NSCLCother, NSCLCsquam, PACA, PRCA, UEC87 NYTRLVLQF CCC, GBC, GC, NSCLCsquam, OSCAR, UEC 88 TYVPSTFLVCCC, GBM, GC, NSCLCsquam, OSCAR, PACA 89 TYVPSTFLVVL GC 90 TDLVQFLLF RCC91 KQQVVKFLI PRCA 92 RALTETIMF UEC 93 TDWSPPPVEF NSCLCsquam 94 THSGGTNLFNSCLCadeno 95 IGLSVVHRF PRCA 96 SHIGVVLAF PRCA 97 TQMFFIHAL PRCA 98LQIPVSPSF MEL 99 ASAALTGFTF OC, PRCA 100 KVWSDVTPLTFCCC, NSCLCsquam, PACA 101 VYAVSSDRF GBM 102 VLASAHILQF NSCLCadeno 103EMFFSPQVF CRC, HCC, NSCLCadeno 104 GYGLTRVQPF NSCLCadeno 105 ITPATALLLNHL 106 LYAFLGSHF RCC 107 FFFKAGFVWR NSCLCsquam, UEC 108 WFFQGAQYW HCC109 AQHSLTQLF GBM 110 VYSNPDLFW NSCLCadeno 111 IRPDYSFQF NSCLCadeno 112LYPDSVFGRLF OC 113 ALMSAFYTF NSCLCsquam 114 KALMSAFYTF CCC 115 IMQGFIRAFNSCLCadeno 116 TYFFVANKY GC 117 RSMEHPGKLLF NSCLCadeno, UEC 118IFLPFFIVF GC 119 VWSCEGCKAF UBC 120 VYAFMNENF RCC 121 RRYFGEKVAL PRCA122 YFLRGRLYW NSCLCsquam, OC 123 FFLQESPVF HCC 124 EYNVFPRTLCRC, GC, NSCLCadeno, NSCLCsquam 125 LYYGSILYI MEL 126 YSLLDPAQFCRC, NSCLCadeno, PACA, PRCA, UEC 127 FLPRAYYRW PRCA 128 AFQNVISSF GC 129IYVSLAHVL GC, NSCLCother, NSCLCsquam, PRCA 130 RPEKVFVF NSCLCadeno 131MHRTWRETF PRCA 132 TFEGATVTL CRC, MEL, NHL 133 FFYVTETTF NHL 134IYSSQLPSF CLL 135 KYKQHFPEI HCC 136 YLKSVQLF NSCLCsquam 137 ALFAVCWAPFNSCLCsquam 138 MMVTVVALF NSCLCsquam, RCC 139 AYAPRGSIYKF GBC, NSCLCadeno140 IFQHFCEEI CLL 141 QYAAAITNGL NSCLCsquam 142 PYWWNANMVF NHL 143KTKRWLWDF NSCLCadeno, NSCLCsquam 144 LFDHGGTVFF GBM 145 MYTIVTPML GC 146NYFLDPVTI BRCA, MEL, NSCLCadeno, NSCLCsquam, OC 147 FPYPSSILSV GBM 148MLPQIPFLLL OC 149 TQFFIPYTI CCC, GC, NSCLCsquam, OC 150 FIPVAWLIF OC 151RRLWAYVTI MEL 152 MHPGVLAAFLF NSCLCadeno 153 AWSPPATLF MEL 154 DYSKQALSLNSCLCadeno 155 PYSIYPHGVTF HCC 156 IYPHGVTFSP GC 157 SIYPHGVTF PRCA 158SYLKDPMIV CLL 159 VFQPNPLF UEC 160 YIANLISCF GBC 161 ILQAPLSVFCLL, GC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, RCC, SCLC162 YYIGIVEEY HCC, NSCLCsquam 163 YYIGIVEEYW GC 164 MFQEMLQRL MEL 165KDQPQVPCVF GC 166 MMALWSLLHL NSCLCsquam 167 LQPPWTTVFGC, MEL, NSCLCadeno, NSCLCsquam 168 LSSPVHLDF CRC, HCC, NHL, NSCLCadeno,OSCAR, RCC, UEC 169 MYDLHHLYL NSCLCsquam 170 IFIPATILL HCC 171 LYTVPFNLINSCLCsquam 172 RYFIAAEKILW HNSCC 173 RYLSVCERL NSCLCsquam 174 TYGEEMPEEIPRCA 175 SYFEYRRLL NSCLCadeno 176 TQAGEYLLF AML 177 KYLITTFSL NSCLCadeno178 AYPQIRCTW AML 179 MYNMVPFF NSCLCadeno 180 IYNKTKMAF SCLC 181IHGIKFHYF GC 182 AQGSGTVTF CLL 183 YQVAKGMEF NSCLCadeno 184 VYVRPRVFNSCLCadeno 185 LYICKVELM GC 186 RRVTWNVLF NSCLCsquam 187 KWFNVRMGFGF NHL188 SLPGSFIYVF NSCLCadeno, RCC 189 FYPDEDDFYF NSCLCother 190 IYIIMOSCWAML 191 MSYSCGLPSL GC, HCC 192 CYSFIHLSF HCC, NHL 193 KYKPVALQCIANSCLCadeno 194 EYFLPSLEII CLL 195 IYNEHGIQQI GBM, NSCLCadeno, NSCLCsquam196 VGRSPVFLF OC 197 YYHSGENLY GC, NSCLCadeno 198 VLAPVSGQFCLL, GBC, GC, MEL, NHL, NSCLCadeno, NSCLCsquam, SCLC 199 MFQFEHIKWNSCLCadeno 200 LYMSVEDFI NSCLCother 201 VFPSVDVSF CRC, GBM 202VYDTMIEKFA GBM, NSCLCadeno, NSCLCother, NSCLCsquam 203 VYPSESTVM GBM 204WQNVTPLTF NSCLCsquam 205 ISWEVVHTVF PACA 206 EVVHTVFLFGBC, MEL, NSCLCadeno, NSCLCsquam, PRCA, SCLC 207 IYKFIMDRFAML, GBC, GC, HCC, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PRCA, UBC208 QYLQQQAKL NSCLCadeno 209 DIYVTGGHLF PRCA 210 EAYSYPPATINSCLCadeno, NSCLCsquam 211 MLYFAPDLIL GC 212 VYFVQYKIM UBC 213 FYNRLTKLFNSCLCadeno 214 YIPMSVMLF NSCLCadeno 215 KASKITFHW GBM 216 RHYHSIEVF HCC217 QRYGFSSVGF HCC 218 FYFYNCSSL UEC 219 KVVSGFYYI GC 220 TYATHVTEI GC221 VFYCLLFVF CCC, NSCLCsquam 222 HYHAESFLF OSCAR 223 KLRALSILF PRCA 224AYLQFLSVL NSCLCother 225 ISMSATEFLL OSCAR 226 TYSTNRTMI CCC 227YLPNPSLNAF CLL, GC, NSCLCadeno, NSCLCsquam 228 VYLRIGGFMEL, NSCLCadeno, NSCLCsquam, OSCAR, PRCA, RCC, SCLC, UBC 229 CAMPVAMEFNHL 230 RWLSKPSLL NHL 231 KYSVAFYSLD GC 232 IWPGFTTSI GC 233 LYSRRGVRTLOC 234 RYKMLIPF PRCA 235 VYISDVSVY NSCLCadeno 236 LHLYCLNTF CCC 237RQGLTVLTW NSCLCadeno 238 YTCSRAVSLF NSCLCadeno 239 IYTFSNVTF GC 240RVHANPLLI HCC 241 QKYYITGEAEGF OSCAR 242 SYTPLLSYI MEL 243 ALFPMGPLTFCLL 244 TYIDTRTVFL HCC 245 VLPLHFLPF RCC 246 KIYTTVLFANI HCC 247VHSYLGSPF RCC 248 CWGPHCFEM NSCLCsquam 249 HQYGGAYNRV OC 250 VYSDRQIYLLHCC 251 DYLLSWLLF GC 252 RYLIIKYPF GBC 253 QYYCLLLIFNSCLCadeno, NSCLCsquam 254 KQHAWLPLTI NHL 255 VYLDEKQHAW CLL, NHL 256QHAWLPLTI NHL 257 MLILFFSTI MEL 258 VCWNPFNNTF CRC 259 FFLFIPFFNSCLCadeno 260 FLFIPFFIIF GC 261 IMFCLKNFWW NSCLCadeno, OC 263 AYVTEFVSLBRCA, GBC 264 AYAIPSASLSW HCC 265 LYQQSDTWSL HCC, NSCLCadeno 266TQIITFESF MEL 267 QHMLPFWTDL OSCAR 268 YQFGWSPNF GC 269 FSFSTSMNEF PRCA270 GTGKLFWVF PACA 271 INGDLVFSF CLL 272 IYFNHRCF NSCLCadeno 273VTMYLPLLL GC, NSCLCadeno, NSCLCsquam, OC, RCC 274 EYSLPVLTFAML, CCC, CLL, CRC, GBC, GBM, GC, HCC, HNSCC, MEL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, RCC, SCLC, UBC, UEC 275PEYSLPVLTF NHL 277 MSAIWISAF OSCAR 278 TYESVVTGFF SCLC 279 KYKNPYGF HCC280 TIYSLEMKMSF NSCLCother, NSCLCsquam 281 MDQNQVVWTF NSCLCadeno 282ASYQQSTSSFF NSCLCadeno 283 SYIVDGKII PRCA 284 QFYSTLPNTI HCC 285YFLPGPHYF RCC 286 HHTQLIFVF GBC, PRCA 287 LVQPQAVLFGBM, GC, NSCLCadeno, SCLC, UEC 288 MGKGSISFLF NSCLCadeno 289 RTLNEIYHWNSCLCadeno, NSCLCsquam 290 VTPKMLISF CCC, NSCLCother 291 YTRLVLQFAML, BRCA, CCC, CLL, CRC, GBC, GBM, GC, HCC, HNSCC,MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 292 KMFPKDFRF CRC 293 MYAYAGWFYCRC 294 KMGRIVDYF CCC, GBC, GC 295 KYNRQSMTL HCC 296 YQRPDLLLF CLL, MEL297 LKSPRLFTF GBC 298 TYETVMTFF GBC, GC, HCC, NHL, UBC 299 FLPALYSLLNSCLCadeno 300 LFALPDFIF NSCLCadeno 301 RTALSSTDTF NHL 302 YQGSLEVLFNSCLCadeno 303 RFLDRGWGF CRC 304 YFGNPQKF GC, PACA 305 RNAFSIYILCRC, NSCLCadeno 306 RYILEPFFI BRCA, CCC, CRC, GBC, GBM,GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 307 RILTEFELL GC308 AAFISVPLLI HCC 309 AFISVPLLI NSCLCadeno, NSCLCother 310 EFINGWYVLHCC 311 IQNAILHLF SCLC 312 YLCMLYALF CCC, GBC, GC, NHL 313 IFMENAFEL HCC315 VYDYIPLLL OC 316 IWAERIMF NSCLCsquam 317 DWIWRILFLV GBM 318VQADAKLLF GC, NSCLCadeno, UBC 319 ATATLHLIF UEC 320 EVYQKIILKF GBC 321VYTVGHNLI HCC 322 SFISPRYSWLF AML, CRC, NSCLCadeno, NSCLCsquam, UBC 323NYSPVTGKF OSCAR 324 RYFVSNIYL GC, NSCLCadeno, NSCLCsquam, UEC 325IFMGAVPTL HNSCC, NSCLCadeno, OC, OSCAR 326 VHMKDFFYFCLL, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, PACA 328 IYLVGGYSW CLL 329YLGKNWSF AML, CCC 330 DYIQMIPEL BRCA, GBC, GC, SCLC 332 VYCSLDKSQFBRCA, CCC, GBC, GC, MEL 333 RYADLLIYTY UEC 334 KVFGSFLTL UEC 335RYQSVIYPF UEC 336 VYSDLHAFY CCC, GBC, HCC, NSCLCadeno, PACA 337SHSDHEFLF UEC 338 VYLTWLPGL CLL, NHL 339 KQVIGIHTFCLL, GBC, GBM, GC, RCC 340 FPPTPPLF AML 341 RYENVSILF GBM, OC 342MYGIVIRTI GC 343 EYQQYHPSL NHL 344 YAYATVLTF GBC, GC, NHL, NSCLCadeno,UBC 345 RYLEEHTEF GBC, NSCLCadeno, OC, UEC 346 TYIDFVPYIGBC, GC, NSCLCadeno, OC, RCC, UEC 347 AWLIVLLFL NHL, UEC 348 RSWENIPVTFCCC 349 IYMTTGVLL GC, NSCLCadeno 350 VYKWTEEKF BRCA, NSCLCother, OSCAR351 GYFGTASLF NSCLCadeno, NSCLCother 352 NAFEAPLTF OC 353 AAFPGAFSF OC354 QYIPTFHVY BRCA, GBM, HCC, HNSCC, NSCLCsquam, OSCAR 355 VYNNNSSRFCCC, CRC, GBC, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, SCLC, UEC 356YSLEHLTQF OC, UEC 357 RALLPSPLF MEL, NHL 358 IYANVTEMLL OSCAR 359TQLPAPLRI UEC 360 LYITKVTTI NSCLCadeno 361 KQPANFIVL GBC 362 NYMDTDNLMFCCC, GBC, GC, HCC, OC, OSCAR, SCLC 363 QYGFNYNKF GC, NSCLCsquam 364KQSQVVFVL NSCLCsquam, OC, PRCA 365 KDLMKAYLF GC, SCLC 366 RLGEFVLLF GC367 HWSHITHLF CCC, CRC, GBM, GC, MEL 368 AYFVAMHLFGBC, GBM, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, SCLC, UEC 369 NFYLFPTTF GC, NSCLCsquam 370 TQMDVKLVFGBC, GC, HCC, NHL, NSCLCadeno, NSCLCsquam, OSCAR, UBC, UEC 371 FRSWAVQTFAML, CRC, GC 372 LYHNWRHAF BRCA, OC, RCC 373 IWDALERTFCLL, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno,NSCLCsquam, OC, OSCAR, PRCA 374 MIFAVVVLF GBC, GBM, GC, MEL, NHL,NSCLCadeno, OC, OSCAR, PRCA 375 YYAADQWVF AML, GBC, GC, HCC, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA 376 KYVGEVFNIAML, CLL, HCC, NHL, NSCLCadeno, NSCLCsquam, RCC 377 SLWREVVTF OC, RCC378 VYAVISNIL GBM, NHL, NSCLCadeno, SCLC 379 KLPTEWNVLCCC, CLL, CRC, GC, HCC, NHL, NSCLCadeno, NSCLCother, OSCAR, RCC, UEC 380FYIRRLPMF NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, RCC 381 IYTDITYSFAML, CCC, GBC, HCC, NHL, NSCLCadeno, NSCLCsquam, OC, OSCAR, RCC 382SYPKELMKF GBC, GC, HNSCC, NSCLCadeno, OC, UEC 383 PYFSPSASFNSCLCadeno, NSCLCother, UBC 385 GYFGNPQKF BRCA, CCC, CRC, GBC, GC,HCC, HNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, OC,OSCAR, PACA, PRCA, RCC, SCLC, UBC, UEC 386 YQSRDYYNFCRC, GBC, GBM, GC, HCC, HNSCC, NSCLCadeno, NSCLCsquam, OC, PRCA, UBC,UEC 387 THAGVRLYF CRC, GBC, GC, HCC, NHL, NSCLCadeno, NSCLCsquam,OC, OSCAR, PRCA, UEC AML: acute myeloid leukemia; BRCA: breast cancer;CCC: cholangiocellular carcinoma; CLL: chronic lymphocytic leukemia;CRC: colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma; GC:gastric cancer; HCC: hepatocellular carcinoma; HNSCC: head and necksquamous cell carcinoma; MEL: melanoma; NHL: non-Hodgkin lymphoma;NSCLCadeno: non-small cell lung cancer adenocarcinoma; NSCLCother: NSCLCsamples that could not unambiguously be assigned to NSCLCadeno orNSCLCsquam; NSCLCsquam: squamous cell non-small cell lung cancer; OC:ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA:prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lungcancer; UBC: urinary bladder carcinoma; UEC: uterine and endometrialcancer.

TABLE 8b  Overview of presentation of selected tumor-associated peptides of the present invention across entities. SEQ IDPeptide Presentation on No. Sequence cancer entities 463 VGGNVTSNFAML, BRCA, GBC, GC, HCC, HNSCC, NSCLCsquam, OC 464 VGGNVTSSFBRCA, GBC, GC, HNSCC, NSCLCadeno, NSCLCsquam, OC AML: acute myeloidleukemia; BRCA: breast cancer; GBC: gallbladder cancer; GC: gastriccancer; HCC: hepatocellular carcinoma; HNSCC: head and neck squamouscell carcinoma; NSCLCadeno: non-small cell lung cancer adenocarcinoma;NSCLCsquam: squamous cell non-small cell lung cancer; OC: ovariancancer;.

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-OptionsInc. (Brea, Calif., USA); Geneticist Inc. (Glendale, Calif., USA);ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd(Glasgow, UK). Total RNA from tumor tissues for RNASeq experiments wasobtained from: Asterand (Detroit, Mich., USA & Royston, Herts, UK);BioCat GmbH (Heidelberg, Germany); BioServe (Beltsville, Md., USA);Geneticist Inc. (Glendale, Calif., USA); Istituto Nazionale Tumori“Pascale” (Naples, Italy); ProteoGenex Inc. (Culver City, Calif., USA);University Hospital Heidelberg (Heidelberg, Germany).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tübingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of theensemble sequence database (Ensembl77). Exon reads are normalized forexon length and alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer areshown in FIGS. 2A-2Q. Expression scores for further exemplary genes areshown in Table 9a and Table 9b.

TABLE 9a Expression scores.Exon Expression in tumor types vs normal tissue panel Seq IDhighly over- very highly over- No Sequence over-expressed (+)expressed (++) expressed (+++) 1 IFPKTGLLII BRCA, CRC, GC,HCC, NSCLCadeno, GBC, HNSCC, MEL, UEC UBC NSCLCsquam, OC, OSCAR, SCLC 2LYAPTILLW GBC CCC, HCC 3 KFLTHDVLTELF MEL GBM, SCLC PRCA 4 MVLQPQPQLFGBC, HCC, NHL, PRCA OC, UBC 5 LQPQPQLFFSF GBC, HCC, NHL, PRCA OC, UBC 6IVTFMNKTLGTF NHL CLL 7 GYPLRGSSI OC UEC 8 IMKPLDQDF BRCA, CRC, GBC,NSCLCadeno PRCA GC, NSCLCsquam, PACA, SCLC, UBC, UEC 9 TYINSLAIL PRCA 10QYPEFSIEL PRCA 11 RAMCAMMSF PRCA 12 KYMSRVLFVY MEL, GBM, HNSCC, SCLCNSCLCother NSCLCadeno, NSCLCsquam, OC, OSCAR, UBC, UEC 13 KYYIATMAL MEL,GBM, HNSCC, SCLC NSCLCother NSCLCadeno, NSCLCsquam, OC, OSCAR, UBC, UEC14 YYIATMALI MEL, GBM, HNSCC, SCLC NSCLCother NSCLCadeno,NSCLCsquam, OC, OSCAR, UBC, UEC 15 FMVIAGMPLF NSCLCadeno, RCC NSCLCsquam16 GYFLAQYLM GBM, MEL PRCA 17 IYPEAIATL NSCLCadeno RCC 18 KYVDINTFRLHCC, RCC CCC, GC, MEL, CRC, GBC, HNSCC, NHL, NSCLCadeno, NSCLCother,OC, PACA, UEC NSCLCsquam, OSCAR, SCLC, UBC 19 ILLCMSLLLF OC BRCA 20ELMAHPFLL OC BRCA 21 LYMRFVNTHF AML 22 VYSSFVFNL GBC, GBM, MEL, CLL NHLOC, UBC 23 VYSSFVFNLF GBC, GBM, MEL, CLL NHL OC, UBC 24 KMLPEASLLIGBC, GBM, MEL, CLL NHL OC, UBC 25 MLPEASLLI GBC, GBM, MEL, CLL NHLOC, UBC 26 TYFFVDNQYW HCC, NHL, RCC CCC, CRC, GBC, NSCLCother,GC, HNSCC, MEL, NSCLCsquam NSCLCadeno, OC, OSCAR, PACA, SCLC, UBC, UEC27 LSCTATPLF NSCLCother, OSCAR HNSCC NSCLCsquam, SCLC 28 FWFDSREISF PRCA29 IYLLLPPVI PRCA 30 RQAYSVYAF PRCA 31 KQMQEFFGL CRC, MEL, PACAGC, NSCLCadeno, HNSCC NSCLCsquam, OSCAR, UBC 32 FYPEVELNF CRC, GC, MEL,HNSCC, PACA NSCLCadeno, NSCLCsquam, OSCAR, UBC 33 FYQPDLKYLSF CLL, GBCNHL 34 LIFALALAAF GBC GC, HNSCC, NSCLCsquam, OSCAR, PACA 35 FSSTLVSLFGBC, SCLC HCC, MEL 36 VYLASVAAF PRCA 37 ISFSDTVNVW BRCA, PRCA MEL 38RYAHTLVTSVLF MEL 39 KTYLPTFETTI UEC RCC 40 NYPEGAAYEF BRCA, OC, UEC 41IYFATQVVF PRCA 42 VYDSIWCNM OC, UEC 43 KYKDHFTEI BRCA, MEL,GBC, HCC, NHL NSCLCadeno, NSCLCsquam, SCLC 44 FYHEDMPLW NHL CLL 45YGQSKPWTF MEL 46 IYPDSIQEL HCC 47 SYLWTDNLQEF GBC, SCLC HNSCC,NSCLCsquam, OSCAR 48 AWSPPATLFLF CCC MEL 49 QYLSIAERAEF UEC 50RYFDENIQKF NSCLCother, HNSCC OSCAR 51 YFDENIQKF NSCLCother, HNSCC OSCAR52 SWHKATFLF AML 53 LFQRVSSVSF MEL 54 SYQEAIQQL PRCA 55 AVLRHLETFGBM, GC, OSCAR AML 56 FYKLIQNGF AML 57 RYLQVVLLY GBM, PACA CRC, GBC, GC58 IYYSHENLI CCC HCC 59 VFPLVTPLL GBM 60 RYSPVKDAW BRCA, HNSCCNSCLCsquam, OSCAR, UBC 61 RIFTARLYF NHL 62 VYIVPVIVL GBM, OC CCC 63LYIDKGQYL MEL 64 QFSHVPLNNF MEL 65 EYLLMIFKLV NHL HNSCC, MEL 66IYKDYYRYNF MEL NHL 67 SYVLQIVAI GBM 68 VYKEDLPQL NHL 69 KWFDSHIPRWBRCA, CLL, NHL, UBC RCC 70 RYTGQWSEW AML UBC 71 RYLPNPSLNAF NSCLCother72 RWLDGSPVTL CLL, NHL 73 YFCSTKGQLF NHL CLL 74 NYVLVPTMF GC UEC 75VYEHNHVSL UBC 76 IYIYPFAHW GBC HCC 77 LYGFFFKI UBC 78 TYSKTIALYGF UBC 79FYIVTRPLAF AML CCC 80 SYATPVDLW GBM, GC AML 81 AYLKLLPMF MEL 82SYLENSASW UEC 83 VLQGEFFLF NHL CLL 84 YTIERYFTL GBC, GC, UEC PACA 85KYLSIPTVFF HCC 86 SYLPTAERL CCC 87 NYTRLVLQF GBC, GC, UEC PACA 88TYVPSTFLV GBC, GC, UEC PACA 89 TYVPSTFLVVL GBC, GC, UEC PACA 90TDLVQFLLF HCC, GBC, GC, HNSCC, MEL NSCLCadeno NSCLCsquam, OC,OSCAR, SCLC, UBC 91 KQQVVKFLI GC, HNSCC, NHL, GBC, OC BRCA, PRCANSCLCsquam, OSCAR, PACA, SCLC, UBC, UEC 92 RALTETIMF OC UEC 93TDWSPPPVEF GC, NSCLCsquam, GBC, HCC, HNSCC, MEL PACA, SCLC, UEC OSCAR 94THSGGTNLF HCC, RCC CCC, CRC, GC, GBC, HNSCC, MEL, NHL, NSCLCother,NSCLCadeno, OC, NSCLCsquam OSCAR, PACA, SCLC, UBC, UEC 95 IGLSVVHRF PRCA96 SHIGVVLAF PRCA 97 TQMFFIHAL PRCA 98 LQIPVSPSF GBC, SCLC HCC, MEL 99ASAALTGFTF PRCA 100 KVWSDVTPLTF HCC, RCC, SCLC BRCA, CCC, CRC,GBC, GC, HNSCC, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, UBC, UEC 101VYAVSSDRF GBM, SCLC 102 VLASAHILQF UBC 103 EMFFSPQVF GC, HNSCC, MEL,BRCA, CCC, CRC, NSCLCadeno, GBC, HCC, OC, UBC NSCLCsquam, OSCAR, PACA,RCC, SCLC 104 GYGLTRVQPF GC, HNSCC, MEL, BRCA, CCC, CRC, NSCLCadeno,GBC, HCC, OC, UBC NSCLCsquam, OSCAR, PACA, RCC, SCLC 105 ITPATALLL NHLCLL 106 LYAFLGSHF BRCA, RCC NSCLCadeno, UEC 107 FFFKAGFVWR HCC, OC, SCLCBRCA, CCC, CRC, GBC, GC, HNSCC, NSCLCadeno, NSCLCsquam, OSCAR, PACA,UBC, UEC 108 WFFQGAQYW HCC, OC BRCA, CCC, CRC, GBC, GC, HNSCC,NSCLCadeno, NSCLCsquam, OSCAR, PACA, UBC, UEC 109 AQHSLTQLF GBM, SCLC110 VYSNPDLFW CLL 111 IRPDYSFQF CLL 112 LYPDSVFGRLF AML, CCC, GC,BRCA, GBC, HCC, HNSCC, MEL, NSCLCadeno, NSCLCother, OC, SCLC, UBCNSCLCsquam, OSCAR, UEC 113 ALMSAFYTF HCC, OC, RCC, BRCA, CCC, CRC, SCLCGBC, GC, HNSCC, NSCLCadeno, NSCLCsquam, OSCAR, PACA, UBC, UEC 114KALMSAFYTF HCC, OC, RCC, BRCA, CCC, CRC, SCLC GBC, GC, HNSCC,NSCLCadeno, NSCLCsquam, OSCAR, PACA, UBC, UEC 115 IMQGFIRAF CCC, HCC, OCNSCLCadeno 116 TYFFVANKY CRC, GC, HNSCC, NSCLCadeno, NSCLCsquam,PACA, UBC OSCAR 117 RSMEHPGKLLF BRCA, OC, UEC 118 IFLPFFIVFBRCA, GBC, HCC, CCC, RCC OSCAR, PRCA, UBC, UEC 119 VWSCEGCKAFBRCA, OC, UEC 120 VYAFMNENF NSCLCsquam, RCC OSCAR 121 RRYFGEKVAL PRCA122 YFLRGRLYW HCC, OC BRCA, CCC, CRC, GBC, GC, HNSCC, NSCLCadeno,NSCLCsquam, OSCAR, PACA, UBC, UEC 123 FFLQESPVF HCC, MEL, PRCA BRCA 124EYNVFPRTL NSCLCadeno, UBC HNSCC, NSCLCsquam, OSCAR 125 LYYGSILYI MEL 126YSLLDPAQF GBC, HCC, HNSCC, CRC, GC, PRCA NSCLCadeno, NSCLCother,OSCAR, SCLC, UBC, UEC 127 FLPRAYYRW PRCA 128 AFQNVISSF NSCLCsquam, UECGC, PACA 129 IYVSLAHVL PRCA 130 RPEKVFVF CCC, CRC, GBM, BRCA, GBC, GC,NSCLCother, HNSCC, MEL, NSCLCsquam, OC, NSCLCadeno, SCLC OSCAR, PACA 131MHRTWRETF PRCA 132 TFEGATVTL NHL CLL 133 FFYVTETTF AML, GC, OC, HCC, NHLOSCAR, SCLC, UEC 134 IYSSQLPSF NHL CLL 135 KYKQHFPEI HCC 136 YLKSVQLFBRCA, MEL AML 137 ALFAVCWAPF NSCLCsquam, RCC OSCAR 138 MMVTVVALFNSCLCsquam, RCC OSCAR 139 AYAPRGSIYKF BRCA, HCC NSCLCadeno, OC 140IFQHFCEEI AML, BRCA, GBC, OC, SCLC HCC, MEL, NSCLCadeno 141 QYAAAITNGLSCLC GBM 142 PYWWNANMVF HCC 143 KTKRWLWDF CCC, CRC, GBM, BRCA, GBC,GC, NSCLCother, HNSCC, MEL, NSCLCsquam, OC NSCLCadeno, OSCAR, PACA 144LFDHGGTVFF PRCA 145 MYTIVTPML GBM OC 146 NYFLDPVTI MEL 147 FPYPSSILSVMEL GBM, SCLC 148 MLPQIPFLLL CRC, HNSCC, BRCA, CCC, GBC, NSCLCsquam, OC,GC, NSCLCadeno, UBC, UEC OSCAR, PACA 149 TQFFIPYTI CRC, HNSCC,BRCA, CCC, GBC, NSCLCsquam, OC, GC, NSCLCadeno, UBC, UEC OSCAR, PACA 150FIPVAWLIF HNSCC, UBC MEL 151 RRLWAYVTI MEL 152 MHPGVLAAFLFNSCLCadeno, UBC HNSCC, NSCLCsquam, OSCAR 153 AWSPPATLF CCC MEL 154DYSKQALSL CCC, GC, HNSCC, NSCLCadeno, NSCLCsquam, NSCLCother, OSCARPACA, UBC, UEC 155 PYSIYPHGVTF CCC HCC 156 IYPHGVTFSP CCC HCC 157SIYPHGVTF CCC HCC 158 SYLKDPMIV GBC, HNSCC, MEL, HCC NSCLCadeno, SCLC159 VFQPNPLF GC, NSCLCsquam, CRC, HNSCC, MEL, PACA OC, OSCAR, UBC 160YIANLISCF SCLC, UEC 161 ILQAPLSVF NHL CLL 162 YYIGIVEEY NSCLCother,HNSCC OSCAR 163 YYIGIVEEYW NSCLCother, HNSCC OSCAR 164 MFQEMLQRLBRCA, GBC, HNSCC, MEL, NSCLCsquam, OC, OSCAR, PACA UBC 165 KDQPQVPCVFBRCA 166 MMALWSLLHL SCLC 167 LQPPWTTVF NHL CLL 168 LSSPVHLDF NHL CLL 169MYDLHHLYL CRC, GBC, BRCA, GC, MEL, HNSCC, NHL, NSCLCadeno, OC,NSCLCsquam, UEC OSCAR, PACA 170 IFIPATILL PRCA HCC 171 LYTVPFNLI HCC MEL172 RYFIAAEKILW OSCAR HNSCC 173 RYLSVCERL HNSCC, NSCLCother NSCLCadeno,NSCLCsquam, OSCAR, SCLC, UEC 174 TYGEEMPEEI NSCLCother, HNSCC, OSCARNSCLCsquam 175 SYFEYRRLL GC, NSCLCadeno, CCC, HNSCC, NSCLCother,NSCLCsquam, PACA, UBC OSCAR 176 TQAGEYLLF AML 177 KYLITTFSL BRCA, GC,CCC, GBC, HNSCC, NHL, NSCLCsquam, OC, NSCLCadeno, UBC, UEC OSCAR, PACA,PRCA, SCLC 178 AYPQIRCTW AML 179 MYNMVPFF MEL 180 IYNKTKMAFHNSCC, MEL, OC, HCC, UBC OSCAR, SCLC 181 IHGIKFHYF PACA, UEC GC 182AQGSGTVTF CLL, NHL 183 YQVAKGMEF AML 184 VYVRPRVF MEL 185 LYICKVELM NHLCLL 186 RRVTWNVLF SCLC GBM 187 KWFNVRMGFG GBC, MEL, OC, HCC, SCLC FUBC, UEC 188 SLPGSFIYVF MEL 189 FYPDEDDFYF GBM, MEL, OC, AML, UECSCLC, UBC 190 IYIIMQSCW AML 191 MSYSCGLPSL HNSCC MEL 192 CYSFIHLSFBRCA, CCC, GC, GBC, NSCLCsquam, HNSCC, NHL, OC, UBC, UEC NSCLCadeno,OSCAR, PACA, PRCA, SCLC 193 KYKPVALQCIA MEL 194 EYFLPSLEII MEL 195IYNEHGIQQI CCC, CRC, GBC, BRCA, MEL, GBM, GC, HNSCC, NSCLCadeno, PACANSCLCother, NSCLCsquam, OC, OSCAR 196 VGRSPVFLF CCC, CRC, GBC,BRCA, MEL, GBM, GC, HNSCC, NSCLCadeno, PACA NSCLCother, NSCLCsquam, OC,OSCAR 197 YYHSGENLY MEL CCC 198 VLAPVSGQF NHL CLL 199 MFQFEHIKW HCC 200LYMSVEDFI GC, HNSCC, MEL, CRC, GBC NSCLCadeno, NSCLCsquam, OC,OSCAR, PACA, UBC 201 VFPSVDVSF GBM 202 VYDTMIEKFA GBM 203 VYPSESTVM GBM204 WQNVTPLTF BRCA GBM, MEL 205 ISWEVVHTVF MEL 206 EVVHTVFLF MEL 207IYKFIMDRF NHL, SCLC, UEC NSCLCadeno, NSCLCsquam, OC 208 QYLQQQAKL NHL,SCLC, UEC NSCLCadeno, NSCLCsquam, OC 209 DIYVTGGHLF BRCA, HNSCCNSCLCsquam, OSCAR, UBC 210 EAYSYPPATI MEL 211 MLYFAPDLIL BRCA UEC 212VYFVQYKIM BRCA, HNSCC, CCC, NHL, NSCLCother, NSCLCadeno NSCLCsquam, OC,OSCAR, PACA, UBC, UEC 213 FYNRLTKLF CRC, GBC, OSCAR, UBCHNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OC, PACA, UEC 214 YIPMSVMLFAML, NSCLCsquam, HNSCC, UBC OSCAR 215 KASKITFHW GBM 216 RHYHSIEVF MEL217 QRYGFSSVGF HCC 218 FYFYNCSSL BRCA, CLL, NHL, RCC UBC 219 KVVSGFYYIBRCA, CCC, NHL HNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam,OSCAR, UBC 220 TYATHVTEI BRCA, CCC, NHL HNSCC, MEL, NSCLCadeno,NSCLCother, NSCLCsquam, OSCAR, UBC 221 VFYCLLFVF BRCA, CCC, NHLHNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, UBC 222 HYHAESFLFOSCAR HNSCC 223 KLRALSILF GBM 224 AYLQFLSVL BRCA, HCC, MEL, PRCAOC, SCLC, UEC 225 ISMSATEFLL NSCLCother 226 TYSTNRTMI AML 227 YLPNPSLNAFNSCLCother 228 VYLRIGGF CCC, HNSCC, OC PACA, UBC, UEC 229 CAMPVAMEFCLL, NHL 230 RWLSKPSLL CLL, NHL 231 KYSVAFYSLD MEL, NSCLCsquam, OC, UECRCC, SCLC 232 IWPGFTTSI GBC, GC, PACA, CRC SCLC, UEC 233 LYSRRGVRTLCCC, OC PACA 234 RYKMLIPF BRCA, CCC, GC, GBC, OC, UBC HNSCC, NHL,NSCLCadeno, NSCLCsquam, OSCAR, PACA, SCLC, UEC 235 VYISDVSVY AML, NHLCLL 236 LHLYCLNTF BRCA UEC 237 RQGLTVLTW AML, BRCA, CRC, NHL GC, MEL,NSCLCadeno, OC, OSCAR, PACA, PRCA, SCLC, UEC 238 YTCSRAVSLF GBC, HCC,SCLC NSCLCother, OC, RCC 239 IYTFSNVTF NHL 240 RVHANPLLI HCC 241QKYYITGEAEGF OC BRCA, UEC 242 SYTPLLSYI SCLC GBM 243 ALFPMGPLTF NHL CLL244 TYIDTRTVFL GC UEC 245 VLPLHFLPF HCC, HNSCC, GBC, MEL, NHLNSCLCsquam, UBC 246 KIYTTVLFANI GBC HCC 247 VHSYLGSPF AML 248 CWGPHCFEMGBM RCC 249 HQYGGAYNRV UEC 250 VYSDRQIYLL BRCA 251 DYLLSWLLF NHL CLL 252RYLIIKYPF AML CCC 253 QYYCLLLIF CCC AML 254 KQHAWLPLTI CLL, NHL 255VYLDEKQHAW CLL, NHL 256 QHAWLPLTI CLL, NHL 257 MLILFFSTI GBC, HCC, BRCAHNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, SCLC,UBC, UEC 258 VCWNPFNNTF NSCLCother, OC, UEC RCC, SCLC 259 FFLFIPFFNSCLCadeno, PRCA OC 260 FLFIPFFIIF NSCLCadeno, PRCA OC 261 IMFCLKNFWWMEL 262 YIMFCLKNF MEL 263 AYVTEFVSL SCLC 264 AYAIPSASLSW MEL 265LYQQSDTWSL CLL 266 TQIITFESF NSCLCadeno, HNSCC NSCLCsquam, OSCAR, UBC267 QHMLPFWTDL BRCA, CCC, GBC NSCLCadeno, NSCLCsquam, OC,OSCAR, UBC, UEC 268 YQFGWSPNF HCC 269 FSFSTSMNEF UEC 270 GTGKLFWVF NHLCLL 271 INGDLVFSF UEC 272 IYFNHRCF CLL 273 VTMYLPLLL MEL 274 EYSLPVLTFGBM 275 PEYSLPVLTF GBM 276 KFLGSKCSF HNSCC, UBC NSCLCsquam, OSCAR 277MSAIWISAF MEL 278 TYESVVTGFF HNSCC, UBC NSCLCsquam, OSCAR 279 KYKNPYGFBRCA, CRC, GC, NSCLCsquam HNSCC, NHL, NSCLCother, OC, OSCAR, SCLC, UEC280 TIYSLEMKMSF NSCLCadeno PRCA 281 MDQNQVVWTF NSCLCother, NSCLCadenoNSCLCsquam 282 ASYQQSTSSFF CLL 283 SYIVDGKII HNSCC, MEL, CCCNSCLCsquam, PACA 284 QFYSTLPNTI NSCLCother, NSCLCadeno NSCLCsquam 285YFLPGPHYF CLL, CRC, GBM, NHL GC, HNSCC, MEL, NSCLCadeno, NSCLCsquam, OC,OSCAR, PACA, PRCA, SCLC 286 HHTQLIFVF MEL, HNSCC NSCLCsquam, OSCAR 287LVQPQAVLF NHL CLL 288 MGKGSISFLF GBM, PACA SCLC 289 RTLNEIYHW NHL 290VTPKMLISF HCC 291 YTRLVLQF GBC, GC, UEC PACA 292 KMFPKDFRF PACA RCC 293MYAYAGWFY HNSCC, NSCLCsquam NSCLCadeno, OSCAR 294 KMGRIVDYF GBC, GC, UECPACA 295 KYNRQSMTL HCC 296 YQRPDLLLF NHL CLL 297 LKSPRLFTF UBC 298TYETVMTFF UBC 299 FLPALYSLL CLL 300 LFALPDFIF CLL 301 RTALSSTDTF CLL 302YQGSLEVLF UEC UBC 303 RFLDRGWGF BRCA, CRC, GBC, CCC GC, HNSCC,NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, UBC 304 YFGNPQKFCCC, GC, HNSCC, OSCAR NSCLCother, NSCLCsquam, PACA, UBC 305 RNAFSIYILHNSCC MEL 306 RYILEPFFI HNSCC, NSCLCsquam NSCLCadeno, OSCAR 307RILTEFELL GBC, GC CRC 308 AAFISVPLLI GBC, GC, PACA, CRC UBC 309AFISVPLLI GBC, GC, PACA, CRC UBC 310 EFINGWYVL NHL CLL 311 IQNAILHLFGBC, HCC, NHL HNSCC, MEL, NSCLCadeno, NSCLCother, PACA, SCLC 312YLCMLYALF GBM OC 313 IFMENAFEL HCC 314 SQHFNLATF AML 315 VYDYIPLLL UECUBC 316 IWAERIMF BRCA PRCA 317 DWIWRILFLV BRCA, MEL, GBC NSCLCsquam 318VQADAKLLF GC, HNSCC, CRC NSCLCsquam, OSCAR, UBC 319 ATATLHLIF GBMGBC, MEL, OC, 320 EVYQKIILKF SCLC 321 VYTVGHNLI HCC 322 SFISPRYSWLF AML323 NYSPVTGKF MEL 324 RYFVSNIYL AML, GBC, MEL, OC 325 IFMGAVPTLHNSCC, OC, OSCAR, PRCA 326 VHMKDFFYF CLL, NHL 327 KWKPSPLLF CCC 328IYLVGGYSW CLL, OC 329 YLGKNWSF AML 330 DYIQMIPEL SCLC 331 EYIDEFQSL SCLC332 VYCSLDKSQF SCLC 333 RYADLLIYTY HNSCC, OC, UBC, UEC 334 KVFGSFLTLNSCLCother, UEC 335 RYQSVIYPF NSCLCother, UEC 336 VYSDLHAFY SCLC 337SHSDHEFLF BRCA, GBC, MEL, NSCLCadeno, NSCLCother, OC, PRCA, UBC, UEC 338VYLTWLPGL CLL, NHL 339 KQVIGIHTF CLL 340 FPPTPPLF AML, CLL, NHL 341RYENVSILF GBM 342 MYGIVIRTI CRC, GBC, GC, PACA 343 EYQQYHPSL CLL, NHL344 YAYATVLTF PRCA 345 RYLEEHTEF UBC 346 TYIDFVPYI MEL, OC, RCC, SCLC347 AWLIVLLFL GBC, NHL, OC BRCA, MEL, NHL, 348 RSWENIPVTF SCLC 349IYMTTGVLL MEL 350 VYKWTEEKF CRC, PACA, SCLC 351 GYFGTASLF NSCLCother 352NAFEAPLTF AML, CLL, CRC, HNSCC, NHL, NSCLCsquam, SCLC 353 AAFPGAFSF GBM354 QYIPTFHVY GBM, HCC, HNSCC, NSCLCadeno, OC, OSCAR, SCLC, UBC 355VYNNNSSRF MEL 356 YSLEHLTQF HCC 357 RALLPSPLF HCC HNSCC, OSCAR, 358IYANVTEMLL UBC 359 TQLPAPLRI GBM 360 LYITKVTTI UBC 361 KQPANFIVLGBC, GC, PACA 362 NYMDTDNLMF GBC, GC, PACA BRCA, HNSCC, 363 QYGFNYNKFMEL, OSCAR 364 KQSQVVFVL MEL 365 KDLMKAYLF PRCA, SCLC 366 RLGEFVLLFHNSCC, NSCLCsquam 367 HWSHITHLF CCC, GBC, GBM, MEL 368 AYFVAMHLFGBM, OC, SCLC HNSCC, MEL, 369 NFYLFPTTF OSCAR 370 TQMDVKLVF CLL 371FRSWAVQTF CRC 372 LYHNWRHAF PRCA 373 IWDALERTF BRCA 374 MIFAVVVLF NHL375 YYAADQWVF NHL 376 KYVGEVFNI PRCA 377 SLWREVVTF SCLC 378 VYAVISNILGBM 379 KLPTEWNVL CLL 380 FYIRRLPMF MEL 381 IYTDITYSF MEL 382 SYPKELMKFUBC 383 PYFSPSASF CRC, GBC, HNSCC, UBC NSCLCadeno, NSCLCsquam, OC,OSCAR, SCLC 384 RTRGWVQTL HCC, CRC, GBC, GC, NSCLCadeno, RCC NSCLCsquam,PACA, SCLC 385 GYFGNPQKF CCC, GC, HNSCC, OSCAR NSCLCother, NSCLCsquam,PACA, UBC 386 YQSRDYYNF BRCA, HCC, PRCA 387 THAGVRLYF NSCLCother,NSCLCsquam, SCLC The table lists for each peptide the tumor types forwhich the exon is very highly over-expressed in tumors compared to apanel of normal tissues (+++), highly over-expressed in tumors comparedto a panel of normal tissues (++) or over-expressed in tumors comparedto a panel of normal tissues (+). The baseline for this score wascalculated from measurements of the following relevant normal tissues:blood cells, blood vessels, brain, heart, liver, lung, adipose tissue,adrenal gland, bile duct, bone marrow, esophagus, eye, gallbladder,head-and-neck, kidney, large intestine, lymph node, pancreas,parathyroid gland, peripheral nerve, peritoneum, pituitary, pleura,skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland,trachea, ureter, urinary bladder. In case expression data for severalsamples of the same tissue type were available, the arithmetic mean ofall respective samples was used for the calculation. AML: acute myeloidleukemia; BRCA: breast cancer; CCC: cholangiocellular carcinoma; CLL:chronic lymphocytic leukemia; CRC: colorectal cancer; GBC: gallbladdercancer; GBM: glioblastoma; GC: gastric cancer; HCC: hepatocellularcarcinoma; HNSCC: head and neck squamous cell carcinoma; MEL: melanoma;NHL: non-Hodgkin lymphoma; NSCLCadeno: non-small cell lung canceradenocarcinoma; NSCLCother: NSCLC samples that could not unambiguouslybe assigned to NSCLCadeno or NSCLCsquam; NSCLCsquam: squamous cellnon-small cell lung cancer; OC: ovarian cancer; OSCAR: esophagealcancer; PACA: pancreatic cancer; PRCA: prostate cancer; RCC: renal cellcarcinoma; SCLC: small cell lung cancer; UBC: urinary bladder carcinoma;UEC: uterine and endometrial cancer.

TABLE 9b Expression scores.Exon Expression in tumor types vs normal tissue panel Seq IDhighly over- very highly over- No Sequence over-expressed (+)expressed (++) expressed (+++) 463 VGGNVTSNF AML, HNSCC,GBC, NSCLCsquam, MEL, NHL, OSCAR OC 464 VGGNVTSSF HNSCC, NHLGBC, NSCLCsquam, OC The table lists for each peptide the tumor types forwhich the exon is very highly over-expressed in tumors compared to apanel of normal tissues (+++), highly over-expressed in tumors comparedto a panel of normal tissues (++) or over-expressed in tumors comparedto a panel of normal tissues (+). The baseline for this score wascalculated from measurements of the following relevant normal tissues:blood cells, blood vessels, brain, heart, liver, lung, adipose tissue,adrenal gland, bile duct, bone marrow, esophagus, eye, gallbladder,head-and-neck, kidney, large intestine, lymph node, pancreas,parathyroid gland, peripheral nerve, peritoneum, pituitary, pleura,skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland,trachea, ureter, urinary bladder. In case expression data for severalsamples of the same tissue type were available, the arithmetic mean ofall respective samples was used for the calculation. AML: acute myeloidleukemia; GBC: gallbladder cancer; HNSCC: head and neck squamous cellcarcinoma; MEL: melanoma; NHL: non-Hodgkin lymphoma; NSCLCsquam:squamous cell non-small cell lung cancer; OC: ovarian cancer; OSCAR:esophageal cancer.

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*24:02 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*24leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 461) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.462), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁵ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oreg., USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In vitro immunogenicity for acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer peptides For tested HLA class I peptides,in vitro immunogenicity could be demonstrated by generation of peptidespecific T-cell lines. Exemplary flow cytometry results afterTUMAP-specific multimer staining for 2 peptides of the invention areshown in FIGS. 3A-3G together with corresponding negative controls.Results for 47 peptides from the invention are summarized in Table 10aand Table 10b.

TABLE 10a in vitro immunogenicity of HLA class Ipeptides of the invention Seq Wells positive ID No Sequence Peptide Code[%] 390 KYKDYFPVI MAGEC2-003 + 392 SYEKVINYL MAGEA9-001 + 393 SYNDALLTFTRPM8-004 +++ 395 NYEDHFPLL MAGEA10-002 ++ 398 GYLQGLVSF KLK4-001 ++ 399VWSNVTPLKF MMP12-014 + 400 RYLEKFYGL MMP12-006 + 402 TYKYVDINTFMMP12-004 + 406 KYLEKYYNL MMP1-001 ++ 408 VWSDVTPLTF MMP11-001 + 409VYTFLSSTL ESR1-006 + 411 VYPPYLNYL PGR-002 ++++ 415 KYEKIFEML CT45-002 +416 VFMKDGFFYF MMP1-002 + 420 VYEKNGYIYF MMP13-001 ++++ 422 VWSDVTPLNFMMP13-002 + 429 YYSKSVGFMQW FAM111B-008 ++ 433 NYTSLLVTW PTP-018 + 434VYDTMIEKF PTP-016 + 436 KYLQVVGMF OXTR-001 + 440 NYGVLHVTF NLRP11-002 +447 EYIRALQQL ASCL1-001 + 448 PFLPPAACFF ASCL1-002 + 450 QYDPTPLTWADAMTS12-002 + 453 YYTVRNFTL PTP-014 + 455 KYLSIPTVF UGT1A3-001 +Exemplary results of in vitro immunogenicity experiments conducted bythe applicant for the peptides of the invention. <20% = +; 20%-49% = ++;50%-69% = +++; >=70% = ++++ 

Table 10b in vitro immunogenicity of HLA class Ipeptides of the invention Wells positive Seq ID No Sequence Peptide Code[%] 1 IFPKTGLLII MAGEA4-008 ++ 5 LQPQPQLFFSF POT-002 ++ 22 VYSSFVFNLNLRP4-006 + 23 VYSSFVFNLF NLRP4-007 ++ 26 TYFFVDNQYW MMP12-020 + 32FYPEVELNF MMP1-009 +++ 38 RYAHTLVTSVLF ITIH6-003 ++ 44 FYHEDMPLWFCRL5-004 + 47 SYLWTDNLQEF DNAH17-003 + 52 SWHKATFLF COL24-002 + 56FYKLIQNGF FLT3-010 + 58 IYYSHENLI F5-005 ++ 63 LYIDKGQYL HMCN1-011 + 76IYIYPFAHW NPFFR2-002 +++ 77 LYGFFFKI BTBD16-002 ++++ 78 TYSKTIALYGFBTBD16-004 ++ 79 FYIVTRPLAF SUCN-002 + 81 AYLKLLPMF SLC5A4-001 + 86SYLPTAERL SYT12-001 + 87 NYTRLVLQF GABRP-004 + 88 TYVPSTFLV GABRP-005 +Exemplary results of in vitro immunogenicity experiments conducted bythe applicant for the peptides of the invention. <20% = +; 20%-49% = ++;50%-69% = +++; >=70% = ++++ 

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiation,and exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006)

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100-fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-β2m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

TABLE 11 MHC class I binding scores. Seq ID Peptide No SequencePeptide Code exchange 1 IFPKTGLLII MAGEA4-008 ++++ 2 LYAPTILLW AFP-001++++ 3 KFLTHDVLTELF TRPM8-009 ++++ 5 LQPQPQLFFSF POT-002 ++++ 6IVTFMNKTLGTF ADAM29-001 + 9 TYINSLAIL TGM4-003 ++++ 10 QYPEFSIELTGM4-001 +++ 11 RAMCAMMSF TGM4-002 ++ 12 KYMSRVLFVY CHRNA9-002 ++ 13KYYIATMAL CHRNA9-003 +++ 14 YYIATMALI CHRNA9-004 ++++ 15 FMVIAGMPLFSLC6A3-001 ++ 16 GYFLAQYLM TRPM8-008 +++ 17 IYPEAIATL SLC6A3-002 ++++ 18KYVDINTFRL MMP12-018 ++++ 20 ELMAHPFLL CYP4Z-002 ++ 21 LYMRFVNTHFSPINK2-002 ++ 22 VYSSFVFNL NLRP4-006 +++ 23 VYSSFVFNLF NLRP4-007 +++ 24KMLPEASLLI NLRP4-004 ++ 25 MLPEASLLI NLRP4-005 ++++ 26 TYFFVDNQYWMMP12-020 ++++ 27 LSCTATPLF KHDC1L-001 +++ 28 FWFDSREISF OR51E2-002 ++++29 IYLLLPPVI OR51E2-004 ++++ 30 RQAYSVYAF SLC45A3-005 ++ 31 KQMQEFFGLMMP1-010 +++ 32 FYPEVELNF MMP1-009 ++++ 33 FYQPDLKYLSF NLRP4-003 ++++ 34LIFALALAAF GAST-001 + 35 FSSTLVSLF MAGEC1-003 ++ 36 VYLASVAAFSLC45A3-006 ++++ 37 ISFSDTVNVW ITIH6-001 + 38 RYAHTLVTSVLF ITIH6-003++++ 39 KTYLPTFETTI ENP-002 ++ 40 NYPEGAAYEF ESR1-008 ++++ 41 IYFATQVVFSLC45A3-004 ++++ 42 VYDSIWCNM SCGB2A1-001 ++++ 43 KYKDHFTEI MAGEB1-001++++ 44 FYHEDMPLW FCRL5-004 ++++ 45 YGQSKPWTF PAX3-001 ++ 46 IYPDSIQELLOC-019 +++ 47 SYLWTDNLQEF DNAH17-003 ++++ 48 AWSPPATLFLF LOXL4-003 ++++49 QYLSIAERAEF MSX-001 ++++ 50 RYFDENIQKF HEPHL-004 ++++ 51 YFDENIQKFHEPHL-006 +++ 52 SWHKATFLF COL24-002 ++++ 53 LFQRVSSVSF HMCN1-010 +++ 54SYQEAIQQL NEFH-003 +++ 55 AVLRHLETF CDK6-006 ++ 56 FYKLIQNGF FLT3-010++++ 58 IYYSHENLI F5-005 ++++ 59 VFPLVTPLL PTP-042 ++++ 60 RYSPVKDAWKLHDC7B-006 ++++ 61 RIFTARLYF AICD-001 +++ 62 VYIVPVIVL OXTR-002 ++++ 63LYIDKGQYL HMCN1-011 ++++ 64 QFSHVPLNNF ALX1-001 ++ 66 IYKDYYRYNFPLA2G2D-001 ++++ 67 SYVLQIVAI PTP-041 +++ 68 VYKEDLPQL EML-002 ++++ 69KWFDSHIPRW ERV-002 ++++ 70 RYTGQWSEW IL9R-001 ++++ 71 RYLPNPSLNAFCYP1A1-002 ++++ 72 RWLDGSPVTL CLEC17-005 ++++ 73 YFCSTKGQLF FCRL2-004++++ 74 NYVLVPTMF CAPN6-003 ++++ 75 VYEHNHVSL BTBD16-006 +++ 76IYIYPFAHW NPFFR2-002 ++++ 77 LYGFFFKI BTBD16-002 ++++ 78 TYSKTIALYGFBTBD16-004 ++++ 79 FYIVTRPLAF SUCN-002 +++ 80 SYATPVDLW CDK6-007 ++++ 81AYLKLLPMF SLC5A4-001 ++++ 82 SYLENSASW DLX5-003 ++++ 83 VLQGEFFLFKBTBD8-005 ++++ 84 YTIERYFTL GABRP-007 ++++ 85 KYLSIPTVFF UGT1A3-002++++ 86 SYLPTAERL SYT12-001 ++++ 87 NYTRLVLQF GABRP-004 ++++ 88TYVPSTFLV GABRP-005 ++++ 89 TYVPSTFLVVL GABRP-006 ++++ 90 TDLVQFLLFMAGEA10-003 ++ 92 RALTETIMF ALP-011 ++ 93 TDWSPPPVEF FAM178B-001 +++ 94THSGGTNLF MMP12-019 ++ 95 IGLSVVHRF OR51E2-003 ++++ 96 SHIGVVLAFOR51E2-005 ++ 98 LQIPVSPSF MAGEC1-004 + 99 ASAALTGFTF SLC45A3-003 ++ 100KVWSDVTPLTF MMP11-023 +++ 101 VYAVSSDRF DCX-001 ++++ 102 VLASAHILQFBTBD16-005 ++ 103 EMFFSPQVF ACTL8-002 ++ 104 GYGLTRVQPF ACTL8-003 ++ 106LYAFLGSHF KISS1 R-003 ++++ 108 WFFQGAQYW MMP11-024 ++ 109 AQHSLTQLFGPC2-002 ++ 110 VYSNPDLFW TRDV3-002 ++++ 111 IRPDYSFQF TRDV3-001 ++ 112LYPDSVFGRLF SMC1B-003 ++++ 113 ALMSAFYTF MMP11-020 ++++ 114 KALMSAFYTFMMP11-022 +++ 115 IMQGFIRAF PAE-002 ++ 116 TYFFVANKY MMP1-011 + 117RSMEHPGKLLF ESR1-010 +++ 118 IFLPFFIVF ADAM18-001 ++++ 119 VWSCEGCKAFESR-001 ++ 120 VYAFMNENF QRFPR-004 ++++ 121 RRYFGEKVAL ANO7-005 ++ 123FFLQESPVF ABCC11-004 ++++ 124 EYNVFPRTL MMP13-004 ++ 125 LYYGSILYIOR9-001 ++++ 126 YSLLDPAQF SOX14-002 +++ 127 FLPRAYYRW ANO7-001 ++ 128AFQNVISSF NMUR2-003 ++++ 129 IYVSLAHVL ANO7-002 ++++ 130 RPEKVFVFCOL11A1-005 ++ 131 MHRTWRETF ANO7-004 ++ 133 FFYVTETTF TERT-003 ++++ 134IYSSQLPSF TFEC-004 ++++ 135 KYKQHFPEI MAGEB17-001 +++ 136 YLKSVQLFRFX8-001 ++ 137 ALFAVCWAPF QRFPR-002 ++ 138 MMVTVVALF QRFPR-003 +++ 139AYAPRGSIYKF HHIPL2-001 ++++ 140 IFQHFCEEI SMC1B-002 ++ 141 QYAAAITNGLSALL3-002 +++ 142 PYWWNANMVF NOTU-001 ++++ 143 KTKRWLWDF COL11A1-004 +++144 LFDHGGTVFF ANO7-003 +++ 145 MYTIVTPML OR1N1-001 ++++ 146 NYFLDPVTITRI-005 +++ 148 MLPQIPFLLL COL10-001 ++ 149 TQFFIPYTI COL10-002 ++ 150FIPVAWLIF MRGPRX4-001 +++ 151 RRLWAYVTI ITIH6-002 + 152 MHPGVLAAFLFMMP13-005 +++ 153 AWSPPATLF LOXL4-002 ++++ 154 DYSKQALSL LAMC2-018 ++155 PYSIYPHGVTF F5-006 ++++ 156 IYPHGVTFSP F5-004 + 157 SIYPHGVTF F5-007++ 158 SYLKDPMIV DDX53-001 +++ 159 VFQPNPLF WISP3-002 ++ 160 YIANLISCFGLYATL3-001 ++ 161 ILQAPLSVF FCRL5-005 ++ 162 YYIGIVEEY HEPHL-007 +++163 YYIGIVEEYW HEPHL-008 ++++ 164 MFQEMLQRL TRIML2-001 + 165 KDQPQVPCVFNAT1-001 + 166 MMALWSLLHL ZAC-001 + 167 LQPPWTTVF FCRL5-006 ++ 168LSSPVHLDF FCRL5-007 ++++ 169 MYDLHHLYL EPY-001 ++++ 170 IFIPATILLACSM1-001 ++++ 171 LYTVPFNLI SLC45A2-006 ++++ 172 RYFIAAEKILW HEPHL-005++++ 173 RYLSVCERL NKX-001 ++++ 174 TYGEEMPEEI DNAH17-004 ++ 175SYFEYRRLL LAMC2-019 ++ 176 TQAGEYLLF FLT3-012 ++++ 177 KYLITTFSLNLRP2-008 ++++ 178 AYPQIRCTW FLT3-009 ++++ 179 MYNMVPFF DCT-002 +++ 180IYNKTKMAF SLCO6-001 ++++ 181 IHGIKFHYF NMUR2-004 +++ 182 AQGSGTVTFFCRL3-004 ++ 183 YQVAKGMEF FLT3-014 + 184 VYVRPRVF HMCN1-013 ++ 185LYICKVELM CTL-001 ++++ 186 RRVTWNVLF BTBD17-003 ++ 187 KWFNVRMGFGFLIN-001 ++ 188 SLPGSFIYVF HMCN1-012 ++ 189 FYPDEDDFYF MYCN-002 +++ 190IYIIMQSCW FLT3-011 +++ 191 MSYSCGLPSL KRT33A-001 +++ 192 CYSFIHLSFNLR-006 ++++ 193 KYKPVALQCIA HMCN1-009 ++ 195 IYNEHGIQQI COL11A1-003++++ 196 VGRSPVFLF COL11A1-006 ++ 198 VLAPVSGQF FCRL5-009 +++ 199MFQFEHIKW FBXW10-001 ++ 200 LYMSVEDFI STK31-001 ++++ 201 VFPSVDVSFPTP-043 ++++ 202 VYDTMIEKFA PTP-044 ++ 203 VYPSESTVM PTP-045 ++ 204WQNVTPLTF MMP16-002 ++ 205 ISWEVVHTVF HMCN1-008 ++++ 206 EVVHTVFLFHMCN1-007 ++ 207 IYKFIMDRF FOXB1-001 ++++ 208 QYLQQQAKL FOXB1-002 ++++209 DIYVTGGHLF KLHDC7B-005 +++ 210 EAYSYPPATI HMCN1-006 +++ 211MLYFAPDLIL PGR-004 ++ 212 VYFVQYKIM IL22RA2-001 + 213 FYNRLTKLF OFCC-001++++ 214 YIPMSVMLF HTR7-002 +++ 215 KASKITFHW PTP-038 ++ 216 RHYHSIEVFLOXL4-004 ++ 217 QRYGFSSVGF RHBG-001 ++++ 218 FYFYNCSSL ERV-001 ++++ 219KVVSGFYYI CCR8-003 + 220 TYATHVTEI CCR8-004 ++++ 222 HYHAESFLF HEPHL-003++++ 223 KLRALSILF PTP-039 + 224 AYLQFLSVL GREB-002 ++++ 225 ISMSATEFLLCYP1A1-001 +++ 226 TYSTNRTMI FLT3-013 ++++ 227 YLPNPSLNAF CYP1A1-003 ++228 VYLRIGGF WNT7A-002 ++ 229 CAMPVAMEF KBTBD8-003 ++ 230 RWLSKPSLLKBTBD8-004 ++++ 231 KYSVAFYSLD LAMA1-008 ++ 232 IWPGFTTSI PIWIL1-003++++ 234 RYKMLIPF NLRP2-010 +++ 235 VYISDVSVY CLECL-003 ++++ 236LHLYCLNTF PGR-003 +++ 238 YTCSRAVSLF OTOG-001 ++ 239 IYTFSNVTF BTN1-003++++ 240 RVHANPLLI APOB-081 + 241 QKYYITGEAEGF ESR1-009 ++++ 242SYTPLLSYI C1orf94-002 ++++ 243 ALFPMGPLTF LILRA4-003 ++ 244 TYIDTRTVFLCAPN6-005 ++++ 245 VLPLHFLPF HBG2-001 ++++ 246 KIYTTVLFANI NPFFR2-003++++ 247 VHSYLGSPF MPL-001 ++ 249 HQYGGAYNRV DLX5-002 ++ 250 VYSDRQIYLLABCC11-006 ++++ 251 DYLLSWLLF CNR2-003 ++++ 252 RYLIIKYPF SUCN-003 +++254 KQHAWLPLTI TCL1A-003 + 255 VYLDEKQHAW TCL1A-005 ++++ 256 QHAWLPLTITCL1A-004 + 258 VCWNPFNNTF RNF183-001 +++ 259 FFLFIPFF ADAM2-001 ++ 263AYVTEFVSL SCN3A-001 ++++ 264 AYAIPSASLSW HMCN1-005 ++ 265 LYQQSDTWSLKIAA140-001 ++ 266 TQIITFESF CSF2-001 ++ 267 QHMLPFWTDL NLRP2-009 +++269 FSFSTSMNEF CAPN6-001 ++ 270 GTGKLFWVF BTL-003 + 271 INGDLVFSFCAPN6-002 ++ 272 IYFNHRCF SFMBT1-001 +++ 273 VTMYLPLLL GPR143-001 ++ 274EYSLPVLTF PTP-037 +++ 275 PEYSLPVLTF PTP-040 ++++ 276 KFLGSKCSF HAS3-003++++ 278 TYESVVTGFF HAS3-004 ++ 279 KYKNPYGF MMP20-001 + 280 TIYSLEMKMSFGLB1L3-001 ++ 281 MDQNQVVWTF ROS-008 +++ 282 ASYQQSTSSFF FAM82A1-001 ++283 SYIVDGKII PSG9-001 + 284 QFYSTLPNTI ROS-009 + 285 YFLPGPHYFSOX30-001 ++++ 288 MGKGSISFLF PCSK1-001 ++ 289 RTLNEIYHW FOXP3-001 ++290 VTPKMLISF OR5H8P-001 + 291 YTRLVLQF GABRP-008 ++ 292 KMFPKDFRFTTLL6-001 ++ 294 KMGRIVDYF GABRP-003 ++++ 295 KYNRQSMTL APOB-080 ++++296 YQRPDLLLF GEN-004 ++ 297 LKSPRLFTF BTBD16-001 ++ 298 TYETVMTFFBTBD16-003 ++++ 299 FLPALYSLL CXCR3-001 +++ 300 LFALPDFIF CXCR3-002 ++302 YQGSLEVLF MROH2A-006 ++ 303 RFLDRGWGF ADAMTS12-005 ++++ 306RYILEPFFI SLC7A11-007 ++++ 307 RILTEFELL TRIM31-001 +++ 308 AAFISVPLLITAS2R38-002 ++++ 309 AFISVPLLI TAS2R38-003 ++++ 310 EFINGWYVL MCOLN2-002++ 311 IQNAILHLF OR51B5-001 ++++ 312 YLCMLYALF KCNK18-001 ++ 313IFMENAFEL APOB-079 ++++ 314 SQHFNLATF DNMT3B-003 ++ 315 VYDYIPLLLMROH2A-005 ++++ 316 IWAERIMF TDRD1-001 +++ 318 VQADAKLLF C20-002 + 319ATATLHLIF PCD-007 + 320 EVYQKIILKF PASD-001 ++++ 321 VYTVGHNLI KLB-003++++ 322 SFISPRYSWLF SPNS3-005 ++++ 323 NYSPVTGKF OTOL-001 ++++ 325IFMGAVPTL LPAR3-001 ++++ 326 VHMKDFFYF DYRK4-001 ++++ 327 KWKPSPLLFGPR126-002 ++++ 328 IYLVGGYSW KLHL14-007 ++++ 329 YLGKNWSF SPNS3-006 ++330 DYIQMIPEL RTL-002 ++++ 331 EYIDEFQSL RTL-003 ++++ 332 VYCSLDKSQFRTL-004 ++++ 333 RYADLLIYTY MYO3B-005 ++++ 334 KVFGSFLTL AGTR2-001 ++335 RYQSVIYPF AGTR2-002 ++++ 336 VYSDLHAFY MANEAL-004 ++++ 337 SHSDHEFLFARSH-001 + 338 VYLTWLPGL IFNLR-001 ++++ 339 KQVIGIHTF SFMBT1-002 + 340FPPTPPLF BCL11A-002 + 341 RYENVSILF ADCY8-001 ++++ 342 MYGIVIRTINPSR-001 ++++ 343 EYQQYHPSL CLEC4C-001 ++++ 344 YAYATVLTF ABCC4-002 ++345 RYLEEHTEF MROH2A-003 ++++ 346 TYIDFVPYI TEX15-001 ++++ 348RSWENIPVTF C18orf54-001 ++ 349 IYMTTGVLL TDRD9-002 ++++ 350 VYKWTEEKFTSPE-001 ++++ 351 GYFGTASLF SLC16A14-001 ++++ 352 NAFEAPLTF BRCA2-004+++ 353 AAFPGAFSF CRB2-002 ++ 354 QYIPTFHVY SLC44A5-003 ++++ 355VYNNNSSRF MYO10-003 ++++ 356 YSLEHLTQF ZCCHC16-001 ++ 357 RALLPSPLFSPATA31D1-001 ++ 358 IYANVTEMLL CYP27C-001 ++++ 359 TQLPAPLRI GPR45-001++ 360 LYITKVTTI FSTL4-002 ++++ 361 KQPANFIVL LOC1001246-001 ++ 362NYMDTDNLMF LOCI001246-002 ++++ 363 QYGFNYNKF PNLD-002 ++++ 365 KDLMKAYLFTXNDC16-006 +++ 366 RLGEFVLLF TGM6-001 +++ 367 HWSHITHLF DPY19L1-003++++ 368 AYFVAMHLF TENM4-007 ++++ 369 NFYLFPTTF PNLD-001 ++++ 370TQMDVKLVF GEN-003 ++ 371 FRSWAVQTF NOS2-002 ++ 372 LYHNWRHAF PDE11-002++++ 373 IWDALERTF ABCC11-005 ++ 375 YYAADQWVF CCR4-004 ++++ 376KYVGEVFNI DMXL1-001 ++++ 377 SLWREVVTF CEP250-004 +++ 378 VYAVISNILTNR-003 ++++ 379 KLPTEWNVL AKAP13-005 ++++ 380 FYIRRLPMF CHRNA6-001 ++++381 IYTDITYSF CHRNA6-002 ++++ 382 SYPKELMKF MROH2A-004 ++++ 383PYFSPSASF SPER-001 ++++ 385 GYFGNPQKF LAMA3-002 ++++ 386 YQSRDYYNFAR-002 ++++ 387 THAGVRLYF NUP155-012 ++ 463 VGGNVTSNF CT45-004 +++ 464VGGNVTSSF CT45-005 +++ Binding of HLA-class I restricted peptides toHLA-A*24 was ranged by peptide exchange yield: >10% = +; >20% = ++; >50= +++; >75% = ++++ 

REFERENCE LIST

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The invention claimed is:
 1. A peptide consisting of the amino acidsequence VGGNVTSSF (SEQ ID NO: 464) in the form of a pharmaceuticallyacceptable salt.
 2. The peptide of claim 1, wherein said peptide has theability to bind to an MHC class-I molecule, and wherein said peptide,when bound to said MHC, is capable of being recognized by CD8 T cells.3. The peptide of claim 1, wherein the pharmaceutically acceptable saltis chloride salt.
 4. The peptide of claim 1, wherein thepharmaceutically acceptable salt is acetate salt.
 5. A compositioncomprising the peptide of claim 1, wherein the composition comprises anadjuvant and a pharmaceutically acceptable carrier.
 6. The compositionof claim 5, wherein the peptide is in the form of a chloride salt. 7.The composition of claim 5, wherein the peptide is in the form of anacetate salt.
 8. The composition of claim 5 wherein the adjuvant isselected from the group consisting of anti-CD40 antibody, imiquimod,resiquimod, GM-CSF, cyclophosphamide, sunitinib, bevacizumab,interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives,poly-(I:C) and derivatives, RNA, sildenafil, particulate formulationswith poly(lactide co-glycolide) (PLG), virosomes, interleukin (IL)-1,IL-2, IL-4, IL-7, IL-12, IL-13, IL-15, IL-21, and IL-23.
 9. Thecomposition of claim 8, wherein the adjuvant is IL-2.
 10. Thecomposition of claim 8, wherein the adjuvant is IL-7.
 11. Thecomposition of claim 8, wherein the adjuvant is IL-12.
 12. Thecomposition of claim 8, wherein the adjuvant is IL-15.
 13. Thecomposition of claim 8, wherein the adjuvant is IL-21.
 14. A pegylatedpeptide consisting of the amino acid sequence of VGGNVTSSF (SEQ ID NO:464) or a pharmaceutically acceptable salt thereof.
 15. The peptide ofclaim 14, wherein the pharmaceutically acceptable salt is chloride salt.16. The peptide of claim 14, wherein the pharmaceutically acceptablesalt is acetate salt.
 17. A composition comprising the pegylated peptideof claim 14 or pharmaceutically acceptable salt thereof, and apharmaceutically acceptable carrier.
 18. A peptide consisting of theamino acid sequence of VGGNVTSSF (SEQ ID NO: 464), wherein at least oneamino acid of the peptide is a D-amino acid.
 19. The peptide in the formof a pharmaceutically acceptable salt of claim 1, wherein said peptideis produced by solid phase peptide synthesis or produced by a yeast cellor bacterial cell expression system.
 20. A composition comprising thepeptide of claim 1, wherein the composition is a pharmaceuticalcomposition and comprises water and a buffer.